Pig model for atherosclerosis

[0001] This application is the U.S. National Stage of International Application No. PCT/DK2008/050055, filed on Mar. 6, 2008, published in English. This application claims priority under 35 U.S.C. §119 or 365 to DK PA 2007 00348, filed Mar. 7, 2007. The entire teachings of the above applications are incorporated herein by reference.

[0002] This application incorporates by reference the Sequence Listing contained in the following ASCII text file:

[0003] a) File name 48651003000SequenceListing.txt; created Oct. 24, 2012, 172 KB in size.

[0004] The present invention relates to a genetically modified pig as a model for studying atherosclerosis, wherein the pig model expresses at least one phenotype associated with said disease. The invention further relates to methods by which the genetically modified pig is produced. In addition, methods for evaluating the response of a therapeutical treatment of atherosclerosis, for screening the efficacy of a pharmaceutical composition, and a method for treatment of human being suffering from atherosclerosis are disclosed.

[0005] Transgenic, non-human animals can be used to understand the action of a single gene or genes in the context of the whole animal and the interrelated phenomena of gene activation, expression, and interaction. The technology has also led to the production of models for various diseases in humans and other animals which contributes significantly to an increased understanding of genetic mechanisms and of genes associated with specific diseases.

[0006] Traditionally, smaller animals such as mice have been used as disease models for human diseases and have been found to be suitable as models for certain diseases. However, their value as animal models for many human diseases is quite limited due to differences in mice compared to humans. Larger transgenic animals are much more suitable than mice for the study of many of the effects and treatments of most human diseases because of their greater similarity to humans in many aspects. Particularly, pigs are believed to be valuable as disease models for human diseases.

[0007] Atherosclerosis is by far the most frequent cause of coronary artery disease (angina pectoris, myocardial infarction and sudden death), carotid artery disease (stroke), and peripheral arterial disease. Atherosclerosis is referred to as ‘hardening of the arteries’ which is caused by the formation of numerous plaques within the arteries.

[0008] It is a chronic inflammatory disease, fueled by high plasma levels of cholesterol-rich lipoproteins, that leads to the development of atherosclerotic plaques of inflammatory cells, debris, and smooth muscle cells in large and medium-sized arteries¹. These lesions by themselves rarely cause symptoms. The mechanical process wherein plaques burst open, known as plaque ruptures, causes the devastating consequences of atherosclerosis. By this process the thrombogenic core of the plaque is exposed to the haemostatic system of the circulating blood and this may elicit an acute flow-limiting superimposed thrombus.

[0009] In most cases of atherosclerosis the genetic component is complex, but in some cases the inheritance of the disease is monogenic. These cases are mostly due to mutations in genes coding for proteins involved in lipoprotein trafficking, and the most severe in humans are caused by homozygous null mutations in the low-density lipoprotein (LDL) receptor (homozygous familial hypercholesterolemia). Even though the disease process in homozygous LDL 10 receptor deficiency is immensely aggressive leading to severe coronary atherosclerosis in childhood, the disease is deemed qualitatively no different from that seen in more slowly developing atherosclerosis. Thus, monogenic causes of atherosclerosis can be used as tools to model atherosclerosis and atherosclerotic complications in genetically modified animal models. Apolipoprotein E (ApoE) and LDL-receptor deficient mice with severe hypercholesterolemia and rapid development of atherosclerosis were created in the early 1990s by homologous recombination in embryonic stem cells^6,7. These mouse models have been instrumental in understanding many aspects of plaque development, but they are limited as models for human atherosclerosis because they lack measurable coronary atherosclerosis and do not develop the most feared complication of atherosclerosis, i.e. atherosclerotic plaque rupture and superimposed thrombosis. In addition—because of their small size—these animals have not aided research on bioimaging of atherosclerosis and percutaneous coronary intervention.

[0010] Similarities in cardiovascular structure and coronary artery distribution with humans make swine an attractive species to explore cardiovascular function and diseases. On conventional diets (˜3% fat, w/w), pigs have low plasma cholesterol levels (˜2 mmol/l), but many strains of pigs are susceptible to hypercholesterolemia and moderate atherosclerosis when fed a diet high in saturated fat and cholesterol, including miniature Yucatan⁸ and Yorkshire farm pigs⁹. Yucatan minipigs are of particular interest as models of human atherosclerosis because their adult weight compares well with that of humans (60-80 kg for males and 50-70 kg for females¹⁰) and thus equipment for imaging and percutaneous coronary intervention can be used directly.

[0011] The most pronounced lesions to date have been described in a progeny of large farm pigs identified in Wisconsin that harbor a single-nucleotide missense mutation in the LDL receptor gene that reduces affinity of the receptor to its ligands¹¹. A colony of these pigs is now maintained in France by Professor Ludovic Drouet, INRA, Jouy en Josas. The pigs develop atherosclerosis in coronary arteries with many aspects of human atherosclerosis including plaque ruptures and superimposed thrombosis. However, hypercholesterolemia is modest on a normal pig diet (total cholesterol 5-8 mM) and atherosclerosis develops only slowly over several years. By the time these pigs have developed atherosclerosis, they are by far too large for most scientific purposes.

[0012] Even though considerable advances in anti-atherosclerotic pharmacological therapy have been achieved in the past decades, atherosclerosis remains one of the leading causes of death and severe disability in Denmark and worldwide². There are at least three parts to an explanation for that.

[0013] First, the conventional population-based risk factor approach recommended in official guidelines is unable to identify those who need treatment on the level of the individual³. Thus, even though we have access to effective preventive treatment we are unable to identify those to treat. This problem could be solved by diagnostic imaging of atherosclerosis, which is becoming theoretically possible with the advent of new high-resolution imaging technology^4,5. However, to develop tracers/contrast agents and imaging sequences that are able to visualize atherosclerotic plaques and atherosclerotic disease activity, we need a human-sized animal model of the disease that can be examined in patient CT, MR and PET-scanners.

[0014] Second, anti-atherosclerotic therapy is effective in preventing atherosclerosis in the long-term, but there is a lack of medical therapy that is effective at rapidly decreasing the risk of thrombotic complications in persons that have established severe atherosclerosis. E.g. in those persons that have identified themselves by presenting symptoms of atherosclerosis and in which maximal treatment is instigated, future events might still occur. Today, the major obstacle of developing such medicine is the lack of an animal model in which plaque rupture and arterial thrombosis occurs.

[0015] Third, the best treatment for coronary events today is primary percutaneous coronary intervention with placement of a stent, but these procedures are subject to complications including stent thrombosis and in-stent restenosis. Most research within this important area is carried out in non-diseased coronary pig arteries, but this approach has obvious limitations.

[0016] For these reasons, a human-sized pig model with severe human-like atherosclerosis, including plaque ruptures and thrombotic complications, is needed more than ever.

[0017] Even though the genes responsible for inherited atherosclerosis or involved in the development of disease have been identified in humans it does not follow that animals transgenic for such mutations display a phenotype comparable to that of the human disease. However, the present invention has surprisingly shown that the genetically modified pig models according of the present invention display the atherosclerosis phenotype.

[0018] The present invention concerns a genetically modified pig model which allows for the study of atherosclerosis.

[0019] Thus, one aspect of the present invention relates to a genetically modified pig as a model for studying atherosclerosis, wherein the pig model expresses at least one phenotype associated with said disease and/or a modified pig comprising at least one mutation in the i) endogenous ApoE gene or part thereof and/or LDL gene or part thereof, and/or ii) endogenous ApoE gene or part thereof and/or iii) endogenous LDL receptor gene or part thereof, transcriptional and/or translational product or part thereof and/or a modified pig comprising at least one human and/or porcine proprotein convertase subtilisin/kexin type 9 (PCSK9) gene or part thereof, transcriptional and/or translational product or part thereof.

[0020] Embodiments for the present invention comprises, mini-pigs for example selected from the group consisting of Goettingen, Yucatan, Bama Xiang Zhu, Wuzhishan and Xi Shuang Banna, including any combination thereof. However, another embodiment relates to pigs that are not a mini-pig, such as the species of Sus domesticus, for example where the pig is selected from the group consisting of Landrace, Yorkshire, Hampshire, Duroc, Chinese Meishan, Berkshire and Piêtrain, including any combination thereof. In a preferred embodiment the pig is a Yucatan minipig.

[0021] Embodiments of the present invention comprise the modified pig, wherein the pig comprises at least one mutation in a ApoE and/or LDL gene or part thereof, transcriptional and/or translational product or part thereof. In another embodiment the modified pig comprises at least one mutation in an endogenous ApoE gene or part thereof, transcriptional and/or translational product or part thereof. In a further embodiment the modified pig comprises at least one mutation in an endogenous LDL gene or part thereof, transcriptional and/or translational product or part thereof. In yet another embodiment the at least one mutation is introduced into the endogenous porcine ApoE or LDL gene by homologous recombination or alternatively by random integration. In yet a further embodiment the at least one mutation is introduced into the endogenous porcine ApoE or LDL gene, transcriptional and/or translational product or part thereof by siRNA. A further embodiment relates to the modified pig, wherein said pig comprises at least one human and/or porcine PCSK9 gene or part thereof, transcriptional and/or translational product or part thereof.

[0022] A second aspect of the present invention relates to a genetically modified porcine blastocyst derived from the genetically modified pig model as disclosed herein and/or a modified porcine blastocyst comprising at least one mutation in the i) endogenous ApoE gene or part thereof and/or LDL gene or part thereof, and/or ii) endogenous ApoE gene or part thereof and/or iii) endogenous LDL receptor gene or part thereof, transcriptional and/or translational product or part thereof and/or a modified blastocyst comprising at least one human and/or porcine PCSK9 gene or part thereof, transcriptional and/or translational product or part thereof.

[0023] A third aspect of the present invention relates to a genetically modified porcine embryo derived from the genetically modified pig model as disclosed herein and/or a modified porcine embryo comprising at least one mutation in the i) endogenous ApoE gene or part thereof and/or LDL gene or part thereof, and/or ii) endogenous ApoE gene or part thereof and/or iii) endogenous LDL receptor gene or part thereof, transcriptional and/or translational product or part thereof and/or a modified embryo comprising at least one human and/or porcine PCSK9 gene or part thereof, transcriptional and/or translational product or part thereof.

[0024] A fourth aspect of the present invention relates to a genetically modified porcine fetus derived from the genetically modified pig model as disclosed herein and/or a modified porcine fetus comprising at least one mutation in the i) endogenous ApoE gene or part thereof and/or LDL gene or part thereof, and/or ii) endogenous ApoE gene or part thereof and/or iii) endogenous LDL receptor gene or part thereof, transcriptional and/or translational product or part thereof and/or a modified fetus comprising at least one human and/or porcine PCSK9 gene or part thereof, transcriptional and/or translational product or part thereof.

[0025] A fifth aspect of the present invention relates to a genetically modified porcine donor cell derived from the genetically modified pig model as disclosed herein and/or a modified porcine donor cell comprising at least one mutation in the i) endogenous ApoE gene or part thereof and/or LDL gene or part thereof, and/or ii) endogenous ApoE gene or part thereof and/or iii) endogenous LDL receptor gene or part thereof, transcriptional and/or translational product or part thereof and/or a modified donor cell comprising at least one human and/or porcine PCSK9 gene or part thereof, transcriptional and/or translational product or part thereof.

[0026] In one embodiment of the present invention the at least one phenotype of the modified pig or pig derived from a modified porcine blastocyst, embryo, fetus and/or donor cell is hypercholesterolemia. The hypercholesterolemia is characterized by an at least 10% increase in total cholesterol level in the plasma as compared to a standard level of the pig. In another embodiment of the present invention the at least one phenotype of the modified pig or pig derived from a modified porcine blastocyst, embryo, fetus and/or donor cell is atherosclerosis. The atherosclerosis is for example determined by intravascular ultrasound, PET scanning, CT scanning and/or MR scanning.

[0027] A sixth aspect of the present invention relates to the genetically modified pig as described herein, porcine blastocyst, embryo, fetus and/or donor cell obtainable by nuclear transfer comprising the steps of

• • [0028] i) establishing at least one oocyte having at least a part of a modified zona pellucida,
  • [0029] ii) separating the oocyte into at least two parts obtaining an oocyte having a nucleus and at least one cytoplast,
  • [0030] iii) establishing a donor cell or cell nucleus with desired genetic properties,
  • [0031] iv) fusing at least one cytoplast with the donor cell or membrane surrounded cell nucleus,
  • [0032] v) obtaining a reconstructed embryo,
  • [0033] vi) activating the reconstructed embryo to form an embryo; culturing said embryo; and
  • [0034] vii) transferring said cultured embryo to a host mammal such that the embryo develops into a genetically modified fetus.
    wherein said genetically modified embryo obtainable by nuclear transfer comprises steps i) to v) and/or vi),
    wherein said genetically modified blastocyst obtainable by nuclear transfer comprises steps i) to vi) and/or vii),
    wherein said genetically modified fetus obtainable by nuclear transfer comprises steps i) to vii).

[0035] A seventh aspect of the present invention pertains to a method for producing a transgenic pig as a model for atherosclerosis, porcine blastocyst, embryo, fetus and/or donor cell comprising:

• • [0036] i) establishing at least one oocyte
  • [0037] ii) separating the oocyte into at least three parts obtaining at least one cytoplast,
  • [0038] iii) establishing a donor cell or cell nucleus having desired genetic properties,
  • [0039] iv) fusing at least one cytoplast with the donor cell or membrane surrounded cell nucleus,
  • [0040] v) obtaining a reconstructed embryo,
  • [0041] vi) activating the reconstructed embryo to form an embryo;
  • [0042] vii) culturing said embryo; and
  • [0043] viii) transferring said cultured embryo to a host mammal such that the embryo develops into a genetically modified fetus,
    wherein said genetically modified embryo obtainable by nuclear transfer comprises steps i) to v) and/or vi),
    wherein said genetically modified blastocyst obtainable by nuclear transfer comprises steps i) to vi) and/or vii),
    wherein said genetically modified fetus obtainable by nuclear transfer comprises steps i) to vii).

[0044] Embodiments of the sixth and seventh aspects comprise one or more of the features as defined in any of the preceding claims, wherein the method for activation of the reconstructed embryo is selected from the group of methods consisting of electric pulse, chemically induced shock, increasing intracellular levels of divalent cations and reducing phosphorylation. Further embodiments of the second and third aspects comprise one or more of the features as defined above, wherein steps iv) and vi) are performed sequentially or simultaneously, and embodiments comprising one or more of the features, wherein the embryo is cultured in vitro. Such embryo may be cultured in sequential culture. The embryo, for example at the blastocyst stage, is cryopreserved prior to transfer to a host mammal.

[0045] For the methods of the present invention embodiments cover pigs, mini-pigs for example selected from the group consisting of Goettingen, Yucatan, Bama Xiang Zhu, Wuzhishan and Xi Shuang Banna, including any combination thereof. However, another embodiment relates to pigs that are not a mini-pig, such as the species of Sus scrofa domesticus, for example where the pig is selected from the group consisting of Landrace, Yorkshire, Hampshire, Duroc, Chinese Meishan, Berkshire and Piêtrain, including any combination thereof. In a preferred embodiment is the Yucatan minipig.

[0046] A further aspect of the present invention pertains to method for evaluating the effect of a therapeutical treatment of atherosclerosis, said method comprising the steps of i) providing the pig model as disclosed herein, ii) treating said pig model with a pharmaceutical composition exerting an effect on said phenotype, and iii) evaluating the effect observed.

[0047] In one embodiment the method further comprises a step of advising on medical treatment based on the afore-mentioned observed effects.

[0048] Yet a further aspect of the present invention relates to a method for screening the efficacy of a pharmaceutical composition for atherosclerosis, said method comprising the steps of i) providing the pig model as disclosed herein, ii) expressing in said pig model said genetic determinant and exerting said phenotype for said disease, iii) administering to said pig model a pharmaceutical composition the efficacy of which is to be evaluated, and iv) evaluating the effect, if any, of the pharmaceutical composition on the phenotype exerted by the genetic determinant when expressed in the pig model.

[0049] Furthermore the present invention in another aspect relates to a method for treatment of a human being suffering from atherosclerosis, said method comprising the initial steps of i) providing the pig model as disclosed herein, ii) expressing in said pig model said genetic determinant and exerting said phenotype for said disease, iii) administering to said pig model a pharmaceutical composition the efficacy of which is to be evaluated, and iv) evaluating the effect observed, and v) treating said human being suffering from atherosclerosis based on the effects observed in the pig model.

[0065] The present invention pertains to a genetically modified pig model, blastocysts, donor cells and/or fetuses for studying atherosclerosis wherein the pig model expresses at least one phenotype associated with atherosclerosis.

[0066] It will be appreciated that the invention does not comprise processes for modifying the genetic identity of pigs which are likely to cause them suffering without any substantial medical benefit to man or animal, or animals resulting from such processes.

[0067] The present invention also relates to genetically modified pig embryos obtainable by the methods described herein.

[0068] The methods for producing the pig model for studying atherosclerosis described herein do not encompass a surgical step performed on the pig.

[0069] The term ‘endogenous’ is used herein to specify a particular gene present naturally in the genome of a particular target cell (for example cells of a pig).

[0070] The term “genetic determinant” is used herein to refer to a single-stranded or double-stranded “polynucleotide molecule” or “nucleic acid” comprising a structural gene of interest. The “genetic determinant” encodes a protein not ordinarily made in appreciable amounts in the target cells. Thus, “genetic determinants” include nucleic acids which are not ordinarily found in the genome of the target cell. “Genetic determinants” also include nucleic acids which are ordinarily found within the genome of the target cell, but is in a form which allows for the expression of proteins which are not ordinarily expressed in the target cells in appreciable amounts. Alternatively, “genetic determinants” may encode a variant or mutant form of a naturally-occurring protein.

[0071] The terms “polynucleotide” and “nucleic acid” are used interchangeably, and, when used in singular or plural, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” specifically includes cDNAs. The term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases, are included within the term “polynucleotides” as defined herein. In general, the term “polynucleotide” embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.

[0072] The terms ‘transgenic’ pig and ‘genetically modified’ pig are used in identical meaning herein.

[0073] The present invention pertains to a modified pig model for studying atherosclerosis, wherein the pig model expresses at least one phenotype associated with atherosclerosis.

Pigs

[0074] The present invention relates to a modified pig as a model for studying atherosclerosis, wherein the pig model expresses at least one phenotype associated with atherosclerosis. The pig of the present invention may be any pig.

[0075] The pig is evolutionary close to humans as compared to for example rodentia. Furthermore, the pig has been widely used in bio-medical research because of the similarities between human and porcine physiology (Douglas, 1972; Book & Bustad, 1974).

[0076] In one embodiment the pig is a wild pig. In another embodiment the pig is the domestic pig, Sus scrofa, such as S. domesticus. In yet another embodiment the invention relates to mini pigs, as well as to inbred pigs. The pig can be selected e.g. from the group consisting of Landrace, Yorkshire, Hampshire, Duroc, Chinese Meishan, Berkshire and Pietrain, such as the group consisting of Landrace, Yorkshire, Hampshire and Duroc, for example the group consisting of Landrace, Duroc and Chinese Meishan, such as the group consisting of Berkshire, Pietrain, Landrace and Chinese Meishan, for example the group consisting of Landrace and Chinese Meishan. In one embodiment, the pig is not a mini-pig. In another embodiment the pig of the present invention is an inbred pig.

[0077] In another embodiment of the present invention the pig is a mini-pig and the mini-pig is preferably selected from the group consisting of Goettingen, Yucatan, Bama Xiang Zhu, Wuzhishan and Xi Shuang Banna. Thus, the present invention relates to any of Goettingen, Yucatan, Bama Xiang Zhu, Wuzhishan and Xi Shuang Banna separately, or in any combination. In a preferred embodiment of the invention the Yucatan mini pig is used.

[0078] Due to its size and weight of about 200 kg the domestic pig is not easily handled in a laboratory setting. A preferred alternative to the domestic pig is the Goettingen (Göttingen) mini-pig that weighs about 30 kg. However, alternatives to the Goettingen minipig is the Yucatan minipig, Preferred embodiments of the present invention comprises Goettingen mini pig, or the Yucatan minipig.

Genetically Modified

[0079] The genetic modifications are introduced in the somatic cell prior to cell nuclear transfer. However, the genetic modification may in another embodiment be introduced during the cell nuclear transfer process, for example by addition of transgenes at different steps of the hand made cloning (HMC) procedure that will then find their way to the genome of the embryo.

[0080] The genetic modifications comprise random integration of a disease causing gene, mutated gene, into the genome of the somatic cell. It could also be random integration of a normal non-mutated gene that will cause a disease when expressed in a specific tissue or at a specific expression level.

[0081] However, the invention also pertains to modified pigs, embryos, donor cells, blastocysts and/or fetuses obtained by transfer of mRNA and/or protein of the genes disclosed herein. Thus, the modification of the pig is in one embodiment does not lead to integration of a transgene into the genome of the pig, embryo, blastocyst and/or fetus.

[0082] The introduced gene or transgene, transcriptional and/or translational product or part thereof may originate from any species, including bacteria, pig, human, mouse, rat, yeast, invertebrates, or plants. Regulatory sequences of the transgene may drive ubiquitous or inducible or tissue- and/or time-specific expression and may also originate from any species including pig, human, mouse, rat, yeast, invertebrates, or plants.

[0083] Importantly, the genetic modification in the somatic cell may be targeted to a specific region in the porcine genome by homologous recombination of a targeting construct or by gene editing procedures. This could be inactivation (e.g. knock-out) of specific genes that will cause a disease or phenotype.

[0084] Homologous recombination occurs between two homologous DNA molecules. It is also called DNA crossover. By homologous recombination, one DNA segment can replace another DNA segment with a similar sequence. The process involve breakage and reunion between the homologous regions of DNA, which is mediated by specialized enzymes. The technique allows replacing one allele with an engineered construct without affecting any other locus in the genome. Using homologous recombination it is possible to direct the insertion of a transgene to a specific known locus of the host cells genome. Knowing the DNA sequence of the target locus, it is possible to replace any gene with a genetically modified DNA construct, thereby either replacing or deleting the target sequence. The technique comprises discovering and isolating the normal gene and then determining its function by replacing it in vivo with a defective copy. This procedure is known as ‘gene knock-out’, which allows for specific gene targeting by taking advantage of homologous recombination. Cloned copies of the target gene are altered to make them nonfunctional and are then introduced into ES cells where they recombine with the homologous gene in the cell's genome, replacing the normal gene with a nonfunctional copy.

[0085] Homologous recombination can similarly be exploited to generate fusion genes or insertion of point mutations in a ‘knock-in’ strategy, in which a targeting vector, comprising a relevant exon of the target locus fused with the cDNA sequence of chromosomal translocation-fusion partner, is transfected into embryonic stem cells, whereby the recombinant sequence is fused to an endogenous gene to generate fusion a gene.

[0086] Another applicable technique to exploits the phenomenon called RNA interference (RNAi), in which 21 nucleotide small interfering RNAs (siRNA) can elicit an effective degradation of specific mRNAs. RNA interference constitutes a new level of gene regulation in eukaryotic cells. It is based on the fact that presence of double stranded RNA in a cell eliminates the expression of a gene of the same sequence, whereas expression of other unrelated genes is left undisturbed. The siRNA stimulates the cellular machinery to cut up other single-stranded RNA having the same sequence as the siRNA. In a preferred embodiment of the present invention, siRNAs are directed towards porcine ApoE, and/or porcine LDL. In one embodiment, the siRNA may be selected from the siRNA sequences, described herein.

[0087] The genetic modifications introduced into the porcine genome prior or during the HMC procedure could also be epigenetic modifications (e.g. methylation of DNA or methylation or acetylation/deacetylation of histones) by incubating somatic cells, oocytes or reconstructed HMC embryos with chemical components such as tricostatin or compounds with similar effect.

[0088] The present invention relates to a modified pig, blastocyst, embryo, fetus and/or donor cell comprising a genetic determinant in the form of at least one mutation in an endogenous, and thus porcine, ApoE gene or part thereof, and/or at least one mutation in an endogenous, and thus porcine, LDL gene or part thereof, and/or in the form of an overexpressed human and/or porcine PCSK9 gene or part thereof, separately or in combination as described in detail herein.

[0089] The present invention also relates to porcine embryos, blastocysts and/or fetuses derived from a modified pig expressing at least one phenotype associated with atherosclerosis.

[0090] It is within the scope of the present invention that the modified pig, blastocyst, embryo, fetus and/or donor cell comprises at least one mutation, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 mutations in the ApoE gene and/or the LDL gene.

[0091] In one embodiment of the present invention the modified pig according to the present invention is mutated in the ApoE gene or part thereof, transcriptional and/or translational product or part thereof (Sus scrofa ApoE gene and protein, SEQ ID NO: 1, SEQ ID NO: 2) and/or the LDL gene or part thereof, transcriptional and/or translational product or part thereof (Sus scrofa LDL gene and protein, SEQ ID NO: 3, SEQ ID NO: 4). The genes are mutated so that the function of the gene is disrupted. It is appreciated that a mutation and/or disrupted function of the gene will result in a reduction in the amount of the transcriptional product or part thereof and/or translational product or part thereof of said gene compared to the amount of transcriptional and/or translational product of the gene in question, here the ApoE gene and/or the LDL gene.

[0092] However, in another embodiment the transgenic pig is transgenic for a combination of mutations, for example at least one mutation in the porcine endogenous ApoE gene and/or at least one mutation in the porcine endogenous LDL gene.

[0093] The mutations introduced into the endogenous porcine ApoE gene and/or the endogenous porcine LDL gene or part thereof may be introduced by any method known to the person skilled in the art. In one embodiment the at least one mutation is introduced by knock out through homologous recombination. However, in another preferred embodiment the at least one mutation is in the form of a reduction in the amount of transcriptional and/or translational product or part thereof of the endogenous ApoE and/or endogenous LDL gene compared to the amount of transcriptional and/or translational product or part thereof of a wild type ApoE gene and/or a LDL gene. A preferred method for reducing the amount of ApoE and/or LDL transcriptional and/or translational product or part thereof is by use of small interfering RNAs (siRNA) directed against the transcriptional products or part thereof of the ApoE gene and/or LDL gene. In a preferred embodiment the transcriptional product or part thereof of the LDL gene is targeted. Non-limiting targets for small interfering RNAs knock down of LDL receptors are shown in Table 1. Each of the targets may be chosen separately, or in any combination. In a preferred embodiment the target is T8. In another embodiment the preferred target is T9.

[0094]

TABLE 1
Targets for shRNA directed
LDL receptor knockdown
	First base
	in pig cDNA
	AF065990
	sequence	Sequence
T1	763	tgtcaaagcggcgagtgca (SEQ ID NO: 5)
T2	889	tcccatatctgcaatgacc (SEQ ID NO: 6)
T3	1150	accctggaccgtagtgagt (SEQ ID NO: 7)
T4	1308	tgacaccattattggcgaa (SEQ ID NO: 8)
T5	1309	gacaccattattggcgaag (SEQ ID NO: 9)
T6	1439	agactctcttccaagagaa (SEQ ID NO: 10)
T7	1553	tgaacggagtggacgtcta (SEQ ID NO: 11)
T8	1814	tcacaggctcggacataca (SEQ ID NO: 12)
T9	858	ccaacgagtgtctggacaa (SEQ ID NO: 13)
T10	1109	cctacctcttcttcaccaa (SEQ ID NO: 14)

[0095] One or more mutations of the ApoE and/or LDL gene may be in coding region of the ApoE and/or the LDL gene, however, one or more mutations of the ApoE and/or the LDL gene may also be determined in at least one regulatory sequence of the ApoE and/or LDL gene. By regulatory sequence is meant sequences that regulate the transcriptional and translational process, for example, promoters, enhancers, sequences that affect polyadenylation, translational or transcriptional start, splicing of transcriptional products. The promoters and enhancers that control the transcription of protein-encoding genes are composed of multiple genetic elements. The cellular machinery is able to gather and integrate the regulatory information conveyed by each element, allowing different genes to evolve distinct, often complex patterns of transcriptional regulation. The ApoE and/or LDL gene may alternatively be mutated in one or more of its exons.

[0096] In one embodiment the endogenous porcine ApoE gene of the genetically modified pig, embryo, blastocyst, donor cell and/or fetus of the present invention is mutated in one or more of its exons, thus disrupting gene function of ApoE. Thus, any of exon 1, exon 2, exon 3, or exon 4 of the porcine ApoE gene may be mutated. Exon 1 is positioned at nucleotide 832-857 of the porcine ApoE gene, exon 2 at nucleotide 1663-1728, exon 3 at nucleotides 2473-2662, exon 4 at nucleotides 3037-3879 of the porcine ApoE gene. In a preferred embodiment the one or more exon of ApoE and/or LDL is mutated by disrupting the exon due to the insertion of a nucleotide construct by homologous recombination and knock-out technology.

[0097] Furthermore, it is appreciated the genetically modified pig, embryo, blastocyst, donor cell and/or fetus of the present invention comprises the transcriptional product or part thereof and/or the translational product or part thereof of the porcine ApoE and/or LDL genes as described above.

[0098] In most cases of atherosclerosis the genetic component is complex, but in some cases the inheritance of the disease is monogenic. These cases are mostly caused by mutations in genes coding for proteins involved in lipoprotein trafficking, and the most severe in humans are caused by mutations affecting LDL receptor-mediated lipoprotein uptake (recessive and autosomal dominant familial hypercholesterolemia). Recently, a gain-of-function mutation in the PCSK9 gene was described as the cause of autosomal dominant familial hypercholesterolemia (17) PCSK9 binds to the LDL receptor leading to its degradation (18). Therefore, gain-of-function mutations in humans and overexpression of PCSK9 transgenes in mice leads to functional LDL receptor deficiency (19). The modified pig, embryo, blastocyst, fetus and/or donor cell comprises at least one human and/or porcine PCSK9 gene or part thereof, transcriptional and/or translational product or part thereof.

[0099] In one embodiment the modified pig, embryo, blastocyst, fetus and/or donor cell comprises at least one porcine PCSK9 gene or part thereof, transcriptional and/or translational product thereof. In a preferred embodiment the modified pig, embryo, blastocyst, fetus and/or donor cell comprises at least one human PCSK9 gene or part thereof, transcriptional and/or translational product thereof.

Sequence Identity

[0100] Functional equivalents and variants are used interchangeably herein. In one preferred embodiment of the invention there is also provided variants of the LDL receptor and/or the apolipoprotein A gene. When being polypeptides, variants are determined on the basis of their degree of identity or their homology with a predetermined amino acid sequence, said predetermined amino acid sequence being the LDL receptor and/or the apolipoprotein A gene products as described herein, or, when the variant is a fragment, a fragment of any of the aforementioned amino acid sequences, respectively.

[0101] Accordingly, variants preferably have at least 91% sequence identity, for example at least 91% sequence identity, such as at least 92% sequence identity, for example at least 93% sequence identity, such as at least 94% sequence identity, for example at least 95% sequence identity, such as at least 96% sequence identity, for example at least 97% sequence identity, such as at least 98% sequence identity, for example 99% sequence identity with the predetermined sequence.

[0102] The following terms are used to describe the sequence relationships between two or more polynucleotides: “predetermined sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity”, and “substantial identity”.

[0103] A “predetermined sequence” is a defined sequence used as a basis for a sequence comparision; a predetermined sequence may be a subset of a larger sequence, for example, as a segment of a full-length DNA or gene sequence given in a sequence listing, such as a polynucleotide sequence of the LDL receptor and/or the apolipoprotein A genes as described herein or may comprise a complete DNA or gene sequence. Generally, a predetermined sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.

[0104] Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a predetermined sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the predetermined sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

[0105] Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.

[0106] The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a predetermined sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the predetermined sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the predetermined sequence over the window of comparison. The predetermined sequence may be a subset of a larger sequence, for example, as a segment of the full-length LDL receptor and/or apolipoprotein A gene polynucleotide sequence illustrated herein.

[0107] Sequence identity is determined in one embodiment by utilising fragments of porcine or human ApoE sequence, or porcine or human LDL peptides, porcine or human PCSK9 peptides comprising at least 25 contiguous amino acids and having an amino acid sequence which is at least 80%, such as 85%, for example 90%, such as 95%, for example 96%, such as 97%, for example 98%, such as 99% identical to the amino acids as described herein, wherein the percent identity is determined with the algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.

Conservative Amino Acid Substitutions:

[0108] Substitutions within the groups of amino acids, shown below, are considered conservative amino acid substitutions. Substitutions between the different groups of amino acids are considered non-conservative amino acid substitutions.

P, A, G, S, T (neutral, weakly hydrophobic)

Q, N, E, D, B, Z (hydrophilic, acid amine)

H, K, R (hydrophilic, basic)

F, Y, W (hydrophobic, aromatic)

L, I, V, M (hydrophobic)

C (cross-link forming)

[0109] By the term “transcriptional or translational products” is meant herein products of gene transcription, such as a RNA transcript, for example an unspliced RNA transcript, a mRNA transcript and said mRNA transcript splicing products, and products of gene translation, such as polypeptide(s) translated from any of the gene mRNA transcripts and various products of post-translational processing of said polypeptides, such as the products of post-translational proteolytic processing of the polypeptide(s) or products of various post-translational modifications of said polypeptide(s).

[0110] As used herein, the term “transcriptional product of the gene” refers to a pre-messenger RNA molecule, pre-mRNA, that contains the same sequence information (albeit that U nucleotides replace T nucleotides) as the gene, or mature messenger RNA molecule, mRNA, which was produced due to splicing of the pre-mRNA, and is a template for translation of genetic information of the gene into a protein.

Phenotypes

[0111] The phenotypes associated with atherosclerosis are many. It is appreciated that the pig model of the present invention expresses at least one phenotype associated with atherosclerosis, such as three, for example four, five, six, seven, eight, nine, ten, eleven, 12, 13, 14, 15, 16, 17, 18, 19 or 20 phenotypes associated with atherosclerosis. Non-limiting examples of said phenotypes are hypercholesterolemia, accumulation of fat, cholesterol and other substances in the walls of arteries, plaque formation, stenosis, blockage of blood flow, plaque rupture, infarction, and/or claudication.

[0112] At least one phenotype associated with atherosclerosis is hypercholesterolemia. Hypercholesterolemia is an increase in cholesterol amount as compared to a standard level in the pig observed before the onset of an increase in cholesterol amount or compared to a standard level determined from a population of pigs. The cholesterol amount is preferably measured in the plasma of pigs. An increase in plasma cholesterol is at least 10% compared to a standard level, such at least 15%, for example 20%, such as 25%, for example 30%, such at least 35%, for example at least 40%, such as at least 45%, for example at least 50%, such at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, such at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such at least 95%, for example at least 100%, such as at least 110%, for example at least 120%, such at least 150%, for example at least 175%, such as at least 200%, for example at least 250%, such as at least 300%, for example at least 350%, such at least 400%, for example at least 450%, such as at least 500 compared to a standard level.

[0113] Thus, a 10% increase in hypercholesterolemia in a pig having a standard level of 2 mmol/l corresponds to an amount of cholesterol of 2.2 mmol/l.

[0114] The phenotypes associated with atherosclerosis comprise the building up of fat, cholesterol and other substances in the walls of arteries and form plaques. Eventually, the plaque deposits can make the artery less flexible. The hardening of the artery may result in decreased blood flow (stenosis) and even blockage of blood flow. Consequently, insufficient blood supply to the organs. The artery may compensate for narrowing of the artery by enlarging the artery which when excessive leads to the formation of an aneurysm. If blood flow in the arteries leading to the heart is reduced, chest pain can occur. Plaques can also break apart (plaque rupture), causing pieces of material to move through the artery, causing the formation of a thrombus that will rapidly slow or stop blood flow, leading to death of the tissues affected by stop of blood flow, also known as infarction. This is a common cause of heart attack and stroke. Blood clots can also form around the plaque deposits. Clots block blood flow. If the clot moves into the heart, lungs, or brain, it can cause a stroke, heart attack, or pulmonary embolism.

[0115] Also claudication due to insufficient blood supply to the legs, typically due to a combination of both stenosis and aneurysmal segments narrowed by clots is an embodiment of the phenotypes associated with atherosclerosis. Another phenotype of the genetically modified pig is hypercholesterolemia in which consistently high levels of low-density lipoprotein (LDL) is observed compared to normal levels, leading to premature atherosclerosis of the coronary arteries. Typically in affected men, heart attacks occur in their 40s to 50s. In humans, hypercholesterolemia is often in the form of familial hypercholesterolemia which is mostly due to mutations in genes coding for proteins involved in lipoprotein trafficking, caused by homozygous null mutations in the LDL receptor.

[0116] A number of tests exist which aids in the diagnosis of atherosclerosis are known to the person skilled in the art such as a cardiac stress test which is a medical test performed to evaluate arterial blood flow to the myocardium (heart muscle) during physical exercise, compared to blood flow while at rest. Also low intensity ultrasound is used to detect blood flow velocity in arteries known as a Doppler study, magnetic resonance arteriography, CT scanning, arteriography using x-ray and special dye to see inside the arteries, intravascular ultrasound and/or ultrasonic duplex scanning are used to diagnose atherosclerosis.

Methods for Producing Pig Model for Studying Atherosclerosis

[0117] The present invention provides improved procedures for cloning pigs by nuclear transfer which refers to the introduction of a full complement of nuclear DNA from one cell to an enucleated cell. The genetically modified pig of the present invention may be produced using any technique in which modified genetic material, transcriptional product and/or translational product or part thereof is transferred from at donor cell to a host cell, such as an enucleated oocyte. A number of techniques exist such as introducing genetic material from a genetically modified somatic cell into an enucleated oocyte by for example microinjection or by nuclear transfer

[0118] In cloning, the transfer of the nucleus of a somatic (body) cell or somatic cell into an egg cell (oocyte) which has had its own nucleus removed (denucleated or enucleated) is called somatic cell nuclear transfer. The new individual will develop from this reconstructed embryo and be genetically identical to the donor of the somatic cell. In the present invention the modified pig model, porcine embryo, blastocyst and/or fetus is obtainable by somatic cell nuclear transfer comprising the steps of a) establishing at least one oocyte having at least a part of a modified zona pellucida, b) separating the oocyte into at least two parts obtaining at least one cytoplast, c) establishing a donor cell or cell nucleus having desired genetic properties, d) fusing at least one cytoplast with the donor cell or membrane surrounded cell nucleus, e) obtaining a reconstructed embryo, f) activating the reconstructed embryo to form an embryo; and g) transferring said cultured embryo to a host mammal such that the embryo develops into a genetically modified fetus, wherein said genetically modified embryo obtainable by nuclear transfer comprises steps a) to e) and/or f), wherein said genetically modified blastocyst obtainable by nuclear transfer comprises steps a) to e) and/or f), wherein said genetically modified fetus obtainable by nuclear transfer comprises steps a) to g).

[0119] The oocyte of b) may in another embodiment be separated into at least three parts obtaining at least two cytoplasts. It is appreciated that the donor cell or cell nucleus of c) harbours genetic determinants for atherosclerosis, for example in the form of modified human or porcine ApoE gene or part thereof and/or modified human or porcine LDL gene or part thereof. The host mammal of g) is in one embodiment a pig, preferably a Yucatan mini pig.

[0120] However, the present invention also relates to a method for producing a transgenic pig, porcine blastocyst, embryo and/or fetus as a model for atherosclerosis comprising the steps of a) establishing at least one oocyte, b) separating the oocyte into at least three parts obtaining at least two cytoplasts, c) establishing a donor cell or cell nucleus having desired genetic properties, d) fusing at least one cytoplast with the donor cell or membrane surrounded cell nucleus, e) obtaining a reconstructed embryo, f) activating the reconstructed embryo to form an embryo; and g) transferring said cultured embryo to a host mammal such that the embryo develops into a genetically modified fetus, wherein said genetically modified embryo obtainable by nuclear transfer comprises steps a) to e) and/or f), wherein said genetically modified blastocyst obtainable by nuclear transfer comprises steps a) to e) and/or f), wherein said genetically modified fetus obtainable by nuclear transfer comprises steps a) to g)

[0121] It is appreciated that the donor cell or cell nucleus of c) harbours genetic determinants for atherosclerosis, for example in the form of modified human or porcine ApoE gene or part thereof and/or modified human or porcine LDL gene or part thereof and/or transcriptional and/or translational products thereof, and/or the insertion of a porcine and/or human PCSK9 gene. The host mammal of g) is in one embodiment a pig, preferably a Yucatan mini pig.

[0122] The various parameters are described in detail below.

Oocyte

[0123] The term ‘oocyte’ according to the present invention means an immature female reproductive cell, one that has not completed the maturing process to form an ovum (gamete). In the present invention an enucleated oocyte is the recipient cell in the nuclear transfer process.

[0124] The oocytes according to the present invention are isolated from oviducts and/or ovaries of a mammal. Normally, oocytes are retrieved from deceased pigs, although they may be isolated also from either oviducts and/or ovaries of live pigs. In one embodiment the oocytes are isolated by oviductal recovery procedures or transvaginal recovery methods. In a preferred embodiment the oocytes are isolated by aspiration. Oocytes are typically matured in a variety of media known to a person skilled in the art prior to enucleation. The oocytes can also be isolated from the ovaries of a recently sacrificed animal or when the ovary has been frozen and/or thawed. Preferably, the oocytes are freshly isolated from the oviducts.

[0125] Oocytes or cytoplasts may also be cryopreserved before use. While it will be appreciated by those skilled in the art that freshly isolated and matured oocytes are preferred, it will also be appreciated that it is possible to cryopreserve the oocytes after harvesting or after maturation. If cryopreserved oocytes are utilised then these must be initially thawed before placing the oocytes in maturation medium. Methods of thawing cryopreserved materials such that they are active after the thawing process are well-known to those of ordinary skill in the art. However, in general, cryopreservation of oocytes and cytoplasts is a very demanding procedure, and it is especially difficult in pigs, because of the above mentioned general fragility of pig oocytes and cytoplasts, and because of the high lipid content that makes them very sensitive to chilling injury (i.e. injury that occurs between +15 and +5° C. during the cooling and warming procedure).

[0126] In another embodiment, mature (metaphase II) oocytes that have been matured in vivo, may be harvested and used in the nuclear transfer methods disclosed herein. Essentially, mature metaphase II oocytes are collected surgically from either nonsuperovulated or superovulated pigs 35 to 48 hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone.

[0127] Where oocytes have been cultured in vitro, cumulus cells that are surrounding the oocytes in vivo may have accumulated may be removed to provide oocytes that are at a more suitable stage of maturation for enucleation. Cumulus cells may be removed by pipetting or vortexing, for example, in the presence of in the range of 0.1 to 5% hyaluronidase, such as in the range of 0.2 to 5% hyaluronidase, for example in the range of 0.5 to 5% hyaluronidase, such as in the range of 0.2 to 3% hyaluronidase, for example in the range of 0.5 to 3% hyaluronidase, such as in the range of 0.5 to 2% hyaluronidase, for example in the range of 0.5 to 1% hyaluronidase, such as 0.5% hyaluronidase.

[0128] The first step in the preferred methods involves the isolation of a recipient oocyte from a suitable pig. In this regard, the oocyte may be obtained from any pig source and at any stage of maturation.

[0129] The stage of maturation of the oocyte at enucleation and nuclear transfer has been reported to be of significance for the success of nuclear transfer methods. Immature (prophase I) oocytes from pig ovaries are often harvested by aspiration. In order to employ techniques such as genetic engineering, nuclear transfer and cloning, such harvested oocytes are preferably matured in vitro before the oocyte cells may be used as recipient cells for nuclear transfer.

[0130] Preferably, successful pig embryo cloning uses the metaphase II stage oocyte as the recipient oocyte because it is believed that at this stage of maturation the oocyte can be or is sufficiently activated to treat the introduced nucleus as if it were a fertilising sperm. However, the present invention relates to any maturation stage of the oocyte which is suitable for carrying out somatic cell nuclear transfer, embryos, blastocysts, and/or transgenic pigs obtainable by the method of somatic cell nuclear transfer of the present invention.

[0131] The in vitro maturation of oocytes usually takes place in a maturation medium until the oocyte has reached the metaphase II stage or has extruded the first polar body. The time it takes for an immature oocyte to reach maturation is called the maturation period.

[0132] In a preferred embodiment of the present invention the oocyte is from sow or gilt, preferably from a sow.

[0133] The donor (somatic cell or nucleus of somatic cell) and recipient (cytoplast) involved in the cell nuclear transfer method according to the present invention is a pig. Likewise, reconstructed embryos may be implanted in a pig according to the present invention. The different pigs suitable as donor, recipient or foster mother are described elsewhere herein.

[0134] The donor pig according to the present invention may be female, or male. The age of the pig can be any age such as an adult, or for example a fetus.

Embryo

[0135] According to the present invention a reconstructed embryo (i.e. single cell embryo) contains the genetic material of the donor cell. Subsequently, the reconstructed embryo divides progressively into a multi-cell embryo after the onset of mitosis. In vitro the onset of mitosis is typically induced by activation as described herein.

[0136] In the present invention the term ‘embryo’ also refers to reconstructed embryos which are embryos formed after the process of nuclear transfer after the onset of mitosis by activation. Reconstructed embryos are cultured in vitro.

[0137] When the embryo contains about 12-16 cells, it is called a “morula”. Subsequently, the embryo divides further and many cells are formed, and a fluid-filled cystic cavity within its center, blastocoele cavity. At this stage, the embryo is called a “blastocyst”. The developmental stage of the “fertilized” oocyte at the time it is ready to implant; formed from the morula and consists of an inner cell mass, an internal cavity, and an outer layer of cells called trophectodermal cells.

[0138] The blastocyst according to the present invention may be implanted into the uterus of a host mammal and continues to grow into a fetus and then an animal. In the methods provided herein for producing genetically modified or transgenic non-human mammal, for cloning a non-human mammal, for culturing a reconstructed embryo, and/or for cryopreservation of a pig embryo, the embryo may be cultured in vitro. The embryo may for example be cultured in sequential culture. It will be appreciated that the embryo may be a normal embryo, or a reconstructed embryo as defined elsewhere herein.

[0139] The present invention thus relates to a modified porcine embryo, blastocyst and/or fetus derived from the genetically modified pig model as disclosed herein and/or the modified porcine embryo comprises at least one modified human ApoE gene or part thereof and/or, human LDL gene or part thereof and/or, porcine APoE gene or part thereof and/or, porcine LDL gene or part thereof, and/or comprises a human PCSK9 gene or part thereof, transcriptional and/or translational product or part thereof.

[0140] It is appreciated that the modified porcine embryo, blastocyst and/or fetus derivable from the modified pig model for studying atherosclerosis, expressing at least one phenotype associated with atherosclerosis may have been the result of the crossing of for example a pig transgenic for at least one ApoE mutation and a pig transgenic for at least one LDL mutation, and/or a pig comprising a human PCSK9 gene.

Cytoplast

[0141] An oocyte or a part of an oocyte from which the nucleus has been removed.

Donor Cell

[0142] By the term ‘donor cell’ of the present invention is meant somatic cell and/or cells derived from the germ line.

[0143] By the term ‘somatic cell’ of the present invention is meant any (body) cell from an animal at any stage of development. For example somatic cells may originate from fetal or adult tissue. Especially preferred somatic cells are those of foetal origin. However, cells from a germ line may also be used. According to the present invention a donor cell is a somatic cell. In another embodiment of the present invention the donor cell is a cell derived from a germ cell line.

[0144] In a preferred embodiment of the present invention the donor cell harbours desired genetic properties. However, the donor cell may harbour desired genetic properties which have been gained by genetic manipulation as described elsewhere herein.

[0145] Somatic cells are selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, and other muscle cells.

[0146] These may be obtained from different organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs.

[0147] The pigs from which the somatic cells may be derived are described elsewhere herein. A preferred embodiment of the invention is the use of somatic cells originating from the same species as the recipient oocyte (cytoplast).

[0148] Preferably, the somatic cells are fibroblast cells as the can be obtained from both developing fetuses and adult animals in large quantities. Fibroblasts may furthermore be easily propagated in vitro. Most preferably, the somatic cells are in vitro cultured fibroblasts of foetal origin.

[0149] In a preferred embodiment the somatic cells are genetically modified. In yet a further preferred embodiment of the present invention the somatic cells are preferably of foetal origin, or for example from adults.

[0150] The present invention thus relates to a modified porcine donor cell derived from the genetically modified pig model as disclosed herein and/or the modified porcine embryo comprises at least one modified human ApoE gene or part thereof and/or, human LDL gene or part thereof and/or, porcine APoE gene or part thereof and/or, porcine LDL gene or part thereof, and/or comprises a human PCSK9 gene or part thereof, transcriptional and/or translational product or part thereof.

[0151] It is appreciated that the modified porcine donor cell from the modified pig model for studying atherosclerosis, expressing at least one phenotype associated with atherosclerosis may have been the result of the crossing of for example a pig transgenic for at least one ApoE mutation and a pig transgenic for at least one LDL mutation, and/or a pig comprising a human PCSK9 gene.

Type of Genetic Modification

[0152] The donor cells may be genetically modified by any of standard method known in the art. The genetic modification may be a modification of the genomic DNA by deletion, insertion, duplication and/or other forms of mutation, including point mutation. The modification may be made in coding sequences and/or non-coding sequences. DNA constructs for insertion may harbour a gene of interest and/or regulatory sequences such as promoters, insulators, enhancers, repressors or ribosomal entry sites. In some embodiments, only one genetic modification is introduced in the genome. In other embodiments, however, the genome may be modified at more than one site. Suitable techniques for genetic modification of mammalian cells, such as fibroblasts, include techniques such as gene addition by nonhomologous recombination, gene replacement by homologous recombination, and gene editing. This may include the use of retroviral insertion, transposon transfer and/or artificial chromosome techniques. Nonhomologous DNA recombination may e.g. be carried out as described in Kragh et al. (2004) Reprod. Fert. Dev. 16:290 or Kragh et al. (2004) Reprod. Fert. Dev. 16:315, Transposon-based gene transfer may be carried out as described in Izsvak et al. (1997) Cell 91:501. Gene replacement by homologous recombination may e.g. involve the techniques described by Urnow et al. (2005) Nature 435:646. Techniques for gene editing have been described in Andersen et al. (2002) J. Mol. Med. 80:770, Liu et al (2002) Gene Ther. 9:118 and Sørensen et al. (2005) J. Mol. Med. 83:39.

[0153] In a preferred embodiment the donor cell is genetically modified by random integration, homologous recombination of the genes disclosed herein into the genome of the donor cell, or the transcriptional product or part of the human PCSK9 gene is modified by RNA interference.

[0154] In a preferred embodiment of the present invention the donor cell is genetically modified (as described in a copending application). The donor cell or nucleus carries a SB tagged genome containing a Flp recombination target site for site specific gene insertion or integration. The SB tagged genome result from the integration of a recombinant target vector comprising a DNA transposon construct and a bicistronic gene cassette comprising (i) a FRT recombination site and (ii) an IRES-driven selection gene. The DNA transposon construct may be any construct in which any DNA transposon is present. In the present invention the DNA transposon construct is the Sleeping Beauty (SB) DNA transposon vector. The FRT recombination site may be embedded in the coding sequence of a selection gene which allows for detecting whether a transposition has occurred. The selection gene of the present invention is not limited to any particular selection gene. In preferred embodiments the selection gene are genes conferring resistance to antibiotics or drugs, such as puromycin, tetracycline, streptomycin or hygromycin resistance genes, or the enhanced green fluorescent protein (eGFP) gene, red fluorescent protein genes or the like. The FRT recombination site may thus be embedded in a SV40 promoter driven fusion variant of the selection gene. However, any promoter suitable for conferring expression of a selection gene may be used according to the present invention. Non-limiting examples of such promoters are CMV (cytomegalovirus) or PGK promoter.

[0155] The IRES-driven selection gene is similarly not limited to any particular selection gene. In preferred embodiments the selection gene are genes conferring resistance to antibiotics or drugs, such as puromycin, tetracycline, streptomycin or hygromycin resistance genes, or the enhanced green fluorescent protein (eGFP) gene, red fluorescent protein genes or the like.

[0156] The recombinant vector construct may also comprise at least one site for Cre recombinase. The at least one site for Cre recombinase may be located as disclosed in the examples herein.

[0157] The donor cell or nucleus may also originate from a genetically modified pig comprising at least one site for integration of at least one transgene. A preferred embodiment is a donor cell or nucleus in the form of a fibroblast, such as a primary fibroblast.

[0158] The present invention also relates to a method for producing a porcine cell comprising a SB tagged genome containing a Flp recombination target site for site-specific gene insertion. The method comprises the steps of

[0159] a) providing a mammalian cell, b) transfecting the cell of a) with a plasmid expressing a transposase and a recombinant target vector comprising a DNA transposon construct and a bicistronic gene cassette comprising (i) a FRT recombination site and ii) an IRES-driven selection gene, c) selecting SB tagged cells.

[0160] As described elsewhere herein the mammalian cell may be any cell. In one embodiment in which the porcine cell is subsequently to be used for producing a genetically modified pig by nuclear transfer according to the hand-made protocol as described herein, the porcine cell is in a preferred embodiment a fibroblast and most preferred a porcine primary fibroblast.

[0161] It is appreciated that a desired transgene may be integrated directly into the at least one site for integration present in the genome of the cell. However, the cell in which the genome carries the at least one site for integration is in another embodiment used as a donor cell for the production of a genetically modified pig by for example microinjection of the donor cell or nucleus thereof into a oocyte or by for example somatic nuclear transfer. In a preferred embodiment the donor cell or the nucleus thereof is used for the production of a genetically modified pig by somatic nuclear transfer using the procedure as described elsewhere herein.

[0162] The transgene or gene of interest to be integrated in the targeted cells of the present invention is not limited to any particular gene. In one embodiment the gene to be integrated is a disease-causing gene which results in the formation of a genetically modified pig displaying a phenotype of interest. According to the present invention the gene of interest to be integrated into the porcine cell is Apolipoptrotein-E (ApoE) and/or LDL receptor.

[0163] The integration of the transgene into the at least one site for integration present in the genome of the cell is employed by transfection into the cell of plasmid DNA containing the gene of interest and also FRT sites, and a plasmid expressing the Flp-recombinase used to support integration at the FRT sites.

Enucleation

[0164] The method of enucleation of an oocyte may be selected from the group of methods consisting of aspiration, physical removal, use of DNA-specific fluorochromes, exposure to ultraviolet light and/or chemically assisted enucleation. In one embodiment the present invention relates to the use of DNA-specific fluorochromes. Enucleation may, however, be performed by exposure with ultraviolet light. In a particular embodiment enucleation is chemically assisted prior to physical removal of the nucleus. Chemically assisted enucleation using for example antineoplastic agents, such as demecolcine (N-deacetyl-N-methyl 1 colchicine), and/or for example etoposide or related agents may be performed prior to enzymatic modification of zona pellucida.

[0165] Chemically assisted enucleation comprises culturing matured COCs in maturation medium as described elsewhere herein supplemented with demecolcine for a particular period of time. In the range of 0.1 μg/ml to 10 μg/ml demecolcine, such as 0.2 μg/ml to 10 μg/ml, for example 0.3 μg/ml to 10 μg/ml, such as 0.25 μg/ml to 5 μg/ml, for example 0.3 μg/ml to 1 μg/ml, such as 0.25 μg/ml to 0.5 μg/ml, for example 0.4 μg/ml demecolcin may be supplemented to the maturation medium. Similarly, maturation medium may be supplemented with etoposide for example in the range of 0.1 μg/ml to 10 μg/ml etoposide, such as 0.2 μg/ml to 10 μg/ml, for example 0.3 μg/ml to 10 μg/ml, such as 0.25 μg/ml to 5 μg/ml, for example 0.3 μg/ml to 1 μg/ml, such as 0.25 μg/ml to 0.5 μg/ml, for example 0.4 μg/ml etoposide may be supplemented to the maturation medium. The time for culturing the COCs in the presence of antineoplastic agents ranges from 10 min to 5 hrs, such as 30 minutes to 5 hrs, for example 10 minutes to 2 hrs, such as 30 min to 2 hrs, for example 10 min to 1.5 hrs, such as 20 min to 3 hrs, for example 10 min to 3 hrs, such as 30 min to 1.5 hrs, for example 45 min.

[0166] In a particular embodiment chemically assisted enucleation is performed using 0.45 μg/ml demecolcine and/or etoposide added to the maturation medium for 45 min.

[0167] In a particular embodiment it is preferred that the enucleation is by physical removal of the nucleus. The physical removal may be by separation for example by bisection of the oocyte into two halves (two parts), one which contains the nucleus and the enucleated oocyte half, known as the cytoplast, removing the nucleated half of the oocyte and selecting the resulting cytoplast for further procedures of the invention. Alternatively the separation is by trisection, resulting in three parts of which two parts are cytoplasts. In another embodiment the oocyte may be separated into four parts, resulting in the production of three cytoplasts. The oocyte may even be separated into five parts by physical removal, resulting in four cytoplasts. Similarly, the oocyte may be separated into six parts, for example seven parts, such as eight parts, for example nine parts, such as ten or more parts.

[0168] The physical separation of the oocyte and subsequent removal of the nucleus-bearing part of the oocyte may be achieved by the use of a microsurgical blade. The oocytes may be screened to identify which oocytes have been successfully enucleated. Oocyte parts that harbour nuclear DNA may be identified by staining with Hoechst fluorochrome, the staining procedure of which is known to a person skilled in the art. Oocyte parts harbouring nuclear DNA are discarded and the enucleated oocytes (cytoplasts) are selected for further procedures.

Zona Pellucida

[0169] Zona pellucida is a thick, transparent, noncellular layer or envelope of uniform thickness surrounding an oocyte

[0170] Generally, an intact zona pellucida is considered to be important in cell nuclear transfer due to a number of parameters. One parameter is to keep the polar body close to the metaphase plate of the oocyte in order to indicate the appropriate site for enucleation. Another parameter relates to the keeping of the donor cell close to the oocyte cytoplast before and during fusion. The zona is also believed to confer protection for the donor cell and cytoplast during fusion. Finally, embryo development after reconstitution and activation is believed to be supported by the zona pellucida.

[0171] Modification of at least a part of the zona pellucida can be performed by a number of methods. For example physical manipulation can be used to modify the zona. But also chemical treatment with agents such as acidic solutions (acidic Tyrode) can be employed. One example of chemical agents that can be employed in the present invention is acidic solutions, for example Tyrode. In a particular embodiment of the invention the zona pellucida is modified by enzymatic digestion. Such enzymatic digestion may be performed by enzymes comprising for example trypsin. Alternatively a specific protease may be used, such as pronase.

[0172] In a preferred embodiment the enzymatic digestion results in at least a partial digestion of a part of zona pellucida which in a preferred embodiment of the present invention means that at least a part of the zona pellucida is being removed, or that the zona pellucida is partly removed. In the present context the zona pellucida is not completely removed.

[0173] According to an especially preferred embodiment of the present invention the partially digested part of zona pellucida is characterized by the zona pellucida still being visible and by the fact that the oocyte has not become misshaped.

[0174] The partial digestion may be achieved by exposure to a protease. In another embodiment of the present invention the partial digestion may be accomplished by the use of a pronase. In yet another embodiment the partial digestion may be achieved by a combination of a protease and pronase.

[0175] In a preferred embodiment the concentration of pronase is in the range of 0.1 mg/ml to 10 mg/ml, such as 0.5 mg/ml to 10 mg/ml, for example 1 mg/ml to 10 mg/ml, such as 1.5 mg/ml to 10 mg/ml, for example 2 mg/ml to 10 mg/ml, such as 2.5 mg/ml to 10 mg/ml, for example 2.75 mg/ml to 10 mg/ml, such as 3 mg/ml to 10 mg/ml, for example 3.25 mg/ml to 10 mg/ml, such as 3.3 mg/ml to 10 mg/ml, for example 3.5 mg/ml to 10 mg/ml.

[0176] A preferred embodiment is a pronase concentration in the range of 2 mg/ml to 5 mg/ml, such as 2.25 mg/ml to 5 mg/ml, for example 2.5 mg/ml to 5 mg/ml, such as 2.75 mg/ml to 5 mg/ml, for example 2.8 mg/ml to 5 mg/ml, such as 2.9 mg/ml to 5 mg/ml, for example 3 mg/ml to 5 mg/ml, such as 3.1 mg/ml to 5 mg/ml, for example 3.2 mg/ml to 5 mg/ml, such as 3.3 mg/ml to 5 mg/ml.

[0177] A particular embodiment of the present invention is a pronase concentration in the range of 1 mg/ml to 4 mg/ml, for example 1 mg/ml to 3.9 mg/ml, such as 1 mg/ml to 3.8 mg/ml, for example 1 mg/ml to 3.7 mg/ml, such as 1 mg/ml to 3.6 mg/ml, for example 1 mg/ml to 3.5 mg/ml such as 1 mg/ml to 3.4 mg/ml, for example 1 mg/ml to 3.3 mg/ml.

[0178] In a preferred embodiment the pronase concentration is in the range of 2.5 mg/ml to 3.5 mg/ml, such as 2.75 mg/ml to 3.5 mg/ml, for example 3 mg/ml to 3.5 mg/ml. In a special embodiment the pronase concentration is 3.3 mg/ml.

[0179] It is clear to the skilled person that the pronase should be dissolved in an appropriate medium, one preferred medium according to the present invention is T33 (Hepes buffered TCM 199 medium containing 33% cattle serum (as described earlier—Vajta, et al., 2003).

[0180] The time of incubation of the oocyte in the pronase solution is in the range of 1 second to 30 seconds, such as 2 seconds to 30 seconds, for example 3 seconds to 30 seconds, such as 4 seconds to 30 seconds, such as 5 seconds to 30 seconds.

[0181] In another embodiment of the present invention the incubation time is in the range of 2 seconds to 15 seconds, such as 2 seconds to 14 seconds, for example 2 seconds to 13 seconds, such as 2 seconds to 12 seconds, for example 2 seconds to 11 seconds, such as 2 seconds to 10 seconds, for example 2 seconds to 9 seconds, such as 2 seconds to 8 seconds, for example 2 seconds to 7 seconds, such as 2 seconds to 6 seconds, for example 2 seconds to 5 seconds.

[0182] In a particular embodiment of the present invention the incubation time is in the range of 3 seconds to 10 seconds, such as 3 seconds to 9 seconds, for example 4 seconds to 10 seconds, such as 3 seconds to 8 seconds, for example 4 seconds to 9 seconds, such as 3 seconds to 7 seconds, for example 4 seconds to 8 seconds, such as 3 seconds to 6 seconds, for example 4 seconds to 7 seconds, such as 3 seconds to 5 seconds, for example 4 seconds to 6 seconds, such as 4 seconds to 5 seconds. An especially preferred incubation time is 5 seconds.

[0183] In a preferred embodiment of the present invention the oocyte is treated for 5 seconds in a 3.3 mg/ml pronase solution at 39° C.

Reconstructed Embryo

[0184] By the term ‘reconstructed embryo’ is meant the cell which is formed by insertion of the donor cell or nucleus of the donor cell into the enucleated oocyte which corresponds to a zygote (during normal fertilisation). However, the term ‘reconstructed embryo’ is also referred to as the ‘reconstituted cell’. In the present invention the donor cell is a somatic cell. However, the donor cell may also be derived from a germ line cell.

Fusion

[0185] The transfer of a donor cell or a membrane surrounded nucleus from a donor cell to at least cytoplast is according to the present invention performed by fusion. In the scenarios described below the term ‘donor cell’ also refers to a membrane surrounded nucleus from a donor cell. Fusion may be achieved by a number of methods.

[0186] Fusion may be between a donor cell and at least one cytoplast, such as between a donor cell and at least two cytoplasts, for example between a donor cell and at least two cytoplasts, such as between a donor cell and at least three cytoplasts, such as between a donor cell and at least four cytoplasts, for example between a donor cell and at least five cytoplasts, such as between a donor cell and at least six cytoplasts, for example between a donor cell and at least seven cytoplasts, such as between a donor cell and at least eight cytoplasts.

[0187] Fusion may be performed according to the listed combinations above simultaneously or sequentially. In one embodiment of the present invention the fusion is performed simultaneously. In another embodiment fusion of the at least one cytoplast and a donor cell is performed sequentially.

[0188] For example fusion may be achieved by chemical fusion, wherein a donor cell and the at least one cytoplast are exposed to fusion promoting agents such as for example proteins, glycoproteins, or carbohydrates, or a combination thereof. A variety of fusion-promoting agents are known for example, polyethylene glycol (PEG), trypsin, dimethylsulfoxide (DMSO), lectins, agglutinin, viruses, and Sendai virus. Preferably phytohemaglutinin (PHA) is used. However mannitol and, or polyvinylalcohol may be used.

[0189] Alternatively, fusion may be accomplished by induction with a direct current (DC) across the fusion plane. Often an alternating current (AC) is employed to align the donor and recipient cell. Electrofusion produces a sufficiently high pulse of electricity which is transiently able to break down the membranes of the cytoplast and the donor cell and to reform the membranes subsequently. As a result small channels will open between the donor cell and the recipient cell. In cases where the membranes of the donor cell and the recipient cell connect the small channels will gradually increase and eventually the two cells will fuse to one cell.

[0190] Alignment of the at least one cytoplast and the donor cell may be performed using alternating current in the range of 0.06 to 0.5 KV/cm, such as 0.1 to 0.4 KV/cm, for example 0.15 to 0.3 KV/cm. In a preferred embodiment alignment of the at least one cytoplast and the donor cell may be performed using alternating current at 0.2 KV/cm.

[0191] Fusion may be induced by the application of direct current across the fusion plane of the at least one cytoplast and the donor cell. Direct current in the range of 0.5 to 5 KV/cm, such as 0.75 to 5 KV/cm, for example 1 to 5 KV/cm, such as 1.5 to 5 KV/cm, for example 2 to 5 KV/cm. Another preferred embodiment of the present invention is the application of direct current in the range of 0.5 to 2 KV/cm. In a further preferred embodiment the direct current may be 2 KV/cm.

[0192] The direct current may preferably be applied for in the range of 1-15 micro seconds, such as 5 to 15 micro seconds, for example 5 to 10 micro seconds. A particular embodiment may be 9 micro seconds.

[0193] In an especially preferred embodiment fusion with direct current may be using a direct current of 2 KV/cm for 9 micro seconds.

[0194] Electrofusion and chemical fusion may however be also be combined.

[0195] Typically electrofusion is performed in fusion chambers as known to the skilled person.

[0196] Fusion may be performed in at least one step, such as in two steps, for example three steps, such as in four steps, for example in five steps, such as six steps, for example seven steps, such as in eight steps.

[0197] Fusion may be performed in for example a first step wherein the at least one cytoplast is fused to the donor cell. A second step of fusion may comprise fusion of the fused pair (cytoplast-donor cell, reconstructed embryo) with at least one cytoplast, such as at least two cytoplasts, for example three cytoplasts, such as four cytoplasts, for example five cytoplasts, such as six cytoplasts, for example seven cytoplasts, such as eight cytoplasts. The second step of fusion with fusion of at least one cytoplast and the fused pair may be performed sequentially or simultaneously. In one embodiment the at least two cytoplasts are fused to the fused pair simultaneously. In another embodiment the at least two cytoplasts are fused to the fused pair sequentially.

[0198] In one embodiment of the invention the second step of fusion may also be an activation step wherein the reconstructed embryo is activated to enter mitosis. As described elsewhere herein.

Activation

[0199] In a preferred embodiment the reconstructed embryo may be allowed to rest prior to activation for a period of time in order to allow for the nucleus of the donor cell to reset its genome and gain toti potency in the novel surroundings of the enucleated cytoplast. The reconstructed embryo may for example rest for one hour prior to activation.

[0200] Preferably, the reconstructed embryo may be activated in order to induce mitosis. Methods for activation may preferably be selected from the group of consisting of electric pulse, chemically induced shock, increasing intracellular levels of divalent cations or reducing phosphorylation. A combination of methods may be preferred for activation.

[0201] In one particular embodiment of the invention the activation and the second step of fusion may be performed simultaneously. However, the activation of the reconstituted embryo and the at least one additional step of fusion between the reconstructed embryo and the at least one cytoplast may be performed sequentially.

[0202] Reducing the phosphorylation of cellular proteins in the reconstructed embryo by known methods such as for example by the addition of kinase inhibitors may activate the reconstituted embryo. A preferred embodiment may involve the use of agents that inhibit protein synthesis, for example cycloheximide. A further preferred embodiment may be using agents that inhibit spindle body formation, for example cytochalasin B.

[0203] In one embodiment of the invention the intracellular levels of divalent cations may be increased. Divalent cations such as for example calcium may be in comprised in the activation medium. Preferably, the cations may enter the reconstructed embryo, particularly upon subjecting the reconstructed embryo to an electric pulse. In a preferred embodiment the electric pulse may cause entering of calcium into the reconstructed embryo.

[0204] The application of an electrical pulse using direct current may be an activation step. However, in a preferred embodiment the electrical pulse applied for activation may also serve as an additional fusion step.

[0205] Prior to applying an electrical pulse using direct current the at least one cytoplast and the at least one reconstructed embryo may be aligned by the application of alternating current. The alternating current may be in the range of the range of 0.06 to 0.5 KV/cm, such as 0.1 to 0.4 KV/cm, for example 0.15 to 0.3 KV/cm. In a preferred embodiment alignment of the at least one cytoplast and the donor cell may be performed using alternating current at 0.2 KV/cm.

[0206] Activation may be induced by the application of direct current across the fusion plane of the at least one cytoplast and the donor cell. Direct current in the range of 0.2 to 5 KV/cm, such as 0.4 to 5 KV/cm, for example 0.5 to 5 KV/cm. Another preferred embodiment of the present invention is the application of direct current in the range of 0.5 to 2 KV/cm. In a further preferred embodiment the direct current may be 0.7 KV/cm.

[0207] The direct current may preferably be applied for in the range of 10 to 200 micro seconds, such as 25 to 150 micro seconds, for example 50 to 100 micro seconds. A particular embodiment may be 80 micro seconds.

[0208] In an especially preferred embodiment fusion with direct current may be using a direct current of 0.7 KV/cm for 80 micro seconds.

[0209] An especially preferred embodiment of activation according to the present invention may be use of an electrical pulse in combination with subjecting the reconstructed embryo to agents that inhibit protein synthesis, spindle body formation, and divalent cations.

[0210] Activation may be performed by any combination of the methods described above.

In Vitro Culture of Embryos

[0211] One aspect of the invention relates to a method of in vitro culturing embryos, whereby the blastocyst rate increased to 25.3%. Thus, a method of culturing a reconstructed embryo is within the scope of the present invention, comprising the steps of a) establishing at least one oocyte having at least a part of zona pellucida, b) separating the oocyte into at least two parts obtaining an oocyte having a nucleus and at least one cytoplast, c) establishing a donor cell or cell nucleus having desired genetic properties, d) fusing at least one cytoplast with the donor cell or membrane surrounded cell nucleus, e) obtaining the reconstructed embryo, f) activating the reconstructed embryo to form an embryo, and e) culturing said embryo.

[0212] Another aspect of the invention relates to a method of cell nuclear transfer in which a step of culturing the embryo is included.

[0213] In a preferred embodiment in relation to the methods described herein embryos are cultured in a sequential set of media. Preferably the blastocysts are grown in traditional medium such as for example NCSU37 or equivalent medium as known to a person skilled in the art, wherein glucose is removed and substituted by other agents. One agent may be pyruvate. Another agent may be lactate. The agents may also be combined and replace glucose in the traditional medium.

[0214] The embryos may be cultured in the substituted media as described above from Day 0 to Day 3, such as from Day 0 to Day 2.

[0215] The pyruvate concentration may range from 0.05 to 1 mM, such as 0.1 to 1 mM, for example 0.125 to 1 mM, such as 0.15 to 1 mM. However the concentration of sodium pyruvate may also range from 0.05 mM to 0.9 mM, such as 0.05 to 0.8 mM, for example 0.05 to 0.7 mM, such as 0.05 to 0.6 mM, for example 0.05 to 0.5 mM, such as 0.05 to 0.4 mM, for example 0.05 to 0.3 mM, such as 0.05 to 0.2 mM. Preferably the concentration ranges between 0.05 to 0.17 mM. A preferred concentration of sodium pyruvate is 0.17 mM.

[0216] The lactate concentration may range from 0.5 to 10 mM, such as 0.75 to 10 mM, for example 1 to 10 mM, such as 1.5 to 10 mM, such as 1.75 to 10 mM, for example 2 to 10 mM, such as 2.5 to 10 mM. However the concentration of sodium lactate may also range from 0.5 mM to 9 mM, such as 0.5 to 8 mM, for example 0.5 to 7 mM, such as 0.5 to 6 mM, for example 0.5 to 5 mM, such as 0.5 to 4 mM, for example 0.5 to 03 mM. Preferably the concentration ranges between 1 to 5 mM, such as 2 to 4 mM, for example 2 to 3 mM. A preferred concentration of sodium lactate is 2.73 mM.

[0217] After the initial glucose-free incubation medium glucose is again replacing the pyruvate and lactate. The embryos may be cultured in the glucose containing medium from Day 4 to Day 3, preferably from Day 3 to Day 7. The glucose concentration may range from 1 to 10 mM, such as 2 to 10 mM, for example 3 to 10 mM, such as 4 to 10 mM, for example 5 to 10 mM. However, the glucose concentration may also range from 1 to 9 mM, such as 2 to 8 mM, for example 3 to 7 mM, such as 4-6 mM. A preferred concentration of glucose according to the present invention is 5.5 mM of glucose.

Organ or Tissue Donation

[0218] In one embodiment, the animals of the invention may be used as a source for organ or tissue donation for humans or other animals, either animals of the same species or animal of other species. Transfer between species is usually termed xenotransplantation. Entire organs that may be transplanted include the heart, kidney, liver, pancreas or lung. Alternatively, parts of organs, such as specific organ tissues may be transplanted or transferred to humans or other animals. In a yet further embodiment, an individual cell or a population of individual cells from an animal of the invention may be transferred to a human being or another animal for therapeutic purposes.

Cryopreservation

[0219] The term ‘cryopreserving’ as used herein can refer to vitrification of an oocyte, cytoplast, a cell, embryo, or pig of the invention. The temperatures employed for cryopreservation is preferably lower than −80 degree C., and more preferably at temperatures lower than −196 degree C. Oocytes, cells and embryos of the invention can be cryopreserved for an indefinite amount of time. It is known that biological materials can be cryopreserved for more than fifty years.

[0220] It is within the scope of the present invention that embryos may be cryopreserved prior to transfer to a host pig when employing methods for producing a genetically engineered or transgenic non-human mammal. Such cryopreservation prior to transfer may be at the blastocyst stage the of embryo development. Vitrification is a form of cryopreservation where living cells are rapidly cooled so that the fluid of the cell does not form into ice. Thus, vitrification relates to the process of cooling where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as (typically) −80 C. or −196 C.

[0221] In particular the invention relates to the vitrification of an oocyte, however, the invention also relates to the vitrification of embryos, preferably embryos at the blastocyst stage. In one embodiment, the embryo is cultured to blastocyst stage prior to vitrification. Especially pig embryos are covered by the present invention. Also vitrified cytoplasts are covered by the present invention, as are cells.

[0222] Yet another aspect of the invention relates to the cryopreservation of a pig embryo derived by a method for cell nuclear transfer as described herein comprising a step of vitrifying a pig embryo. A further aspect of the invention relates to pig embryos obtained, or obtainable by the methods provided herein.

Mitochondria

[0223] Cells of the tissue of the genetically modified non-human mammals and/or non-human embryos obtainable by the present invention may harbour mitochondria of different maternal sources. In a preferred embodiment the non-human mammals and/or non-human embryos may harbour mitochondria from only one maternal source, However, in another preferred embodiment the non-human mammals and/or non-human embryos may harbour mitochondria from at least two maternal sources, such as three maternal sources, for example four maternal sources, such as five maternal sources, for example six maternal sources, such as seven maternal sources, for example eight maternal sources, such as nine maternal sources, for example ten maternal sources. The probability of having a specific number of maternal sources can be calculated based on the observed types of mitochondria.

Evaluation of Treatment and Methods for Diagnosis

[0224] The present invention offers a method for screening the efficacy of a pharmaceutical composition, wherein the method comprises the steps of i) providing the pig model of the present invention, ii) expressing in said pig model the genetic determinant and exerting said phenotype for said disease, iii) administering to the pig model a pharmaceutical composition the efficacy of which is to be evaluated, and iv) evaluating the effect, if any, of the pharmaceutical composition on the phenotype exerted by the genetic determinant when expressed in the pig model. In one preferred embodiment the preclinical testing of drugs targeting plaque stability and superimposed thrombosis is within the scope of the present invention.

[0225] Furthermore, within the scope of the present invention is a method for evaluating the response of a therapeutical treatment of atherosclerosis, wherein the method comprises the steps of i) providing the pig model of the present invention, ii) treating said pig model with a pharmaceutical composition exerting an effect on said phenotype, and iii) evaluating the effect observed. Based on the evaluation one could further advise on the treatment based on the observed effects.

[0226] In addition, the present invention relates to a method for treatment of a human being suffering from atherosclerosis, wherein the method comprises the initial steps of i) providing the pig model of the present invention, ii) expressing in said pig model said genetic determinant and exerting said phenotype for said disease, iii) administering to said pig model a pharmaceutical composition the efficacy of which is to be evaluated, and v) evaluating the effect observed, and v) treating said human being suffering from atherosclerosis based on the effects observed in the pig model. In a preferred embodiment the treatment comprises treating a human being suffering from familial hypercholesterolemia.

[0227] It is therefore appreciated that the pig model according to the present invention may also receive medicaments for diseases other than atherosclerosis in order to test the combined effect of a drug for atherosclerosis and other drugs administered to the pig.

[0228] Furthermore, the pig model of the present invention also allows for the development of bioimaging technology for diagnosis of atherosclerosis. Thus, the present invention offers a method for evaluating a bioimaging technology for the diagnosis of atheroclerosis, wherein the method comprises the steps of i) providing the pig model of the present invention, ii) expressing in said pig model the genetic determinant and exerting said phenotype for said disease, iii) bioimaging the pig model and iv) evaluating the result, if any, of the bioimaging technology of the phenotype exerted by the genetic determinant when expressed in the pig model.

[0229] The genetically modified pig of the present invention may also be used in order to improve catheter-based therapies.

[0230] Lipoprotein metabolism in pigs is reasonably well described. The distribution and composition of plasma lipoproteins in swine are similar to those in humans, and LDL is the major cholesterol transporting lipoprotein in both species.¹² Like humans, all VLDL particles secreted by the liver contain the long non-edited form of apoB100 rather than the truncated apoB48 form, which is dominant in mice¹³.

[0231] Apolipoprotein B-containing lipoproteins are cleared by hepatic uptake via 1) the LDL receptor that binds to apolipoprotein E in IDL particles and to apolipoprotein B100 in LDL particles¹⁴, and 2) the LDL receptor-related protein (LRP) that binds to apolipoprotein E in apoB48-containing chylomicron remnants¹⁵. The relative importance of these pathways varies between species. In mice deficiency of apolipoprotein E has a more severe atherosclerotic phenotype than LDL receptor deficiency, whereas the opposite is true in humans. The impact of apoE and LDL receptor deficiency on lipid metabolism in pigs is not known.

ApoE Knockout Pig by Homologous Recombination

[0232] The pig apolipoprotein E gene (GenBank accession no. U70240) has been sequenced.^11,16 A targeting vector construct containing the gene and downstream non-coding sequence is created with a promoter-less neomycin resistance gene cassette inserted into one of the exons to disrupt gene function. The targeting vector is linearized and transfected into Yucatan fetal fibroblasts, isolated from new born Yucatan ear biopsies. Plasmids encoding zinc finger nucleases constructed to recognize and cleave a site in the targeted genomic sequence may be used to increase efficiency of homologous recombination. Fibroblasts are cultured in the presence of G418. Resistant clones are screened for homologous recombination by PCR. Yucatan minipigs with knockout of one or both apoE alleles are created from recombinant cells by “hand-made” cloning.

1. Cloning of Targeting Constructs

[0233] A 1.6 kb fragment containing the 5″-end of the ApoE gene, a 3.2 kb fragment containing the entire ApoE gene, and a 6.1 kb fragment containing the 3″-end of the ApoE gene were all amplified by PCR using genomic DNA extracted from fibroblasts isolated from newborn Yucatan minipigs as template (see below). Upon subcloning and sequencing, parts of the resulting PCR products were used for further cloning into the targeting vector pKO Scrambler NTKV-1903 (Stratagene) comprising a neomycin resistance gene.

SEQ ID NO: 15: 1.6 kb ApoE fragment (5′-end of ApoE)—primers used for amplification are underlined:

[0234]

5′-
gcctgggaatgagtgccagctcctccagttccacgtggcctcaccacacacctcaactctgagtctgggagtcgtgtaac
agggctgctgggggatgggggggtgcagtcagcgctcaccaatctgtcacagaagttaactggaactgttctttgttctatc
cccggatgatggggttaaatgcaaccattttccccgtcttagtggaccgagaaacaatgttcagagaggctaggtcatttg
ctcaaggtcacacagctgacaacccgcagagcctggattcaggcctggaggctttggttccagagttcacagtccgaac
caggcgacgggacaggaacactcccaggcctgtggaaggcgcggtatgcaggccgcgagctcctggaatgcgcaa
ggcttatgtgggggcagagagctgcatcctcattgcacaaatcaggaaagcggctcagagaagcactcagatgtgccc
aaggtcacggccctcgagagggagtgagggttaaaactctgtggtgcaacggaaacgaatccaactgggaaccatga
ggctgtgggttggatccccggcctcgctcaatgggttaaggatccagcacggcgctgccgtgagctgtggtgtaggtcgc
agacgaggcttggatcccacttggctgtggctgtggctgtggctgtggtgtaggcccgcagctgtaactgtaattcgacccc
tagcctgggaacctccacaagccacgggtgtggccctaaaaagcaaaaaaacgaaagcaaaaagaacactctcaa
agcctaaactttcagcaaaaagaacactctcaaagcctaaactttgagcagatgccttacaccgcccccacgcctctcat
cccctttctgtctgggcctccagctcccttcccccttaacccagaaatcccagacctcagacccaggatttcgagtccccag
ccttgccccaattctattcatccaagcacaggacaagagagaggcagggccgggccttctggtcctgctccttctccctgc
ccagcccacccccaccagtggcatggagaaaggctcgggagttactgggtgagagacacctctttccatgggggctgg
gagtaagggggggggtgataggctgccaagccccacccctcccctcccctcccctccccctccctgctgtgtgaaaggg
gaggccagcccacctcgtgacccgacgggggctggcccagctggccccagttctggaggagtgggcggggcgggg
ggagccctataattggccgaatctgggctccctgaatcctactcagccccggaggaggaaggaggaaggaggagga
ggaagcaaccggtgaggagcagacctgggggcacagagatgggctcggggcttcggtgtgggagggtgggctgtag
ggggaggaggaaatgacctggccccccggggccaccaccgaggcaggagttggggatgaggctagagcccaggg
actggacctagaaggagggtgggcagcaggaggaggttatccgccttggctggaaggggaggtcagggaagcagc
gggacctgtaggaagaaccagacgagccagagccgacgaattgtactggc-3′

SEQ ID NO: 16: 3.2 kb ApoE fragment (entire ApoE gene)—primers used for amplification are underlined:

[0235]

5′-
gcccagctggccccagttctggaggagtgggcggggcggggggagccctataattggccgaatctgggctccctgaat
cctactcagccccggaggaggaaggaggaaggaggaggaggaagcaaccggtgaggagcagacctgggggcac
agagatgggctcggggcttcggtgtgggggggtgggctgtagggggaggaggaaatgacctggccccccggggcca
ccaccgaggcaggagttggggatgaggctagagcccagggactggacctagaaggagggtgggcagcaggagga
ggttatccgccttggctggaaggggaggtcagggaagcagcgggacctgtaggaagaaccagacgagccagagcc
gacgaattgtactggcaggtatggcgcatctactcaagttttgagcacactaagagctccatcgaggagacccaggggt
ggcggcgaccaggggtgacctcgaccgggctggcggcagggtagctagagcgttggtggaaggacatgtaaatgag
gattaaattagggaatgagtggaaaacagggtttagatgtgaagttggagcttggaatgtgaaggtaccaggaagaacg
tgagcttggagcccagaaagcaaggctggggctcacatgggactccagggtggagggggtggggggcgacgtgggt
ggaatttgaaccctgggagagagggaaggcttttggccgcagccgacctggggatggggagataggagaagacaat
gagggaattacacggacaatggaaaggatctgctcgggaaatatctgcttggattaggctgatgcagataagggggtgc
aaggcttggaaggctgtgactggacagggctgggctctgggtgggaggagcgagccccgccgctgttgagtgacaattt
ctccctcctgcaggttggccaatcgcaagccagaagatgagggttctgtgggttgctttggtggtaaccctcctcgcaggta
tgggggtggggcttgctcaggttccctgcccctcccccatccccggctgtacccggtgcccctccttcatccctgggtctcttc
tgctggtctctcttccccttgaggagaggcctagatgtgaggcctctctggcactccttgcttctgaacagctcgttttactctct
gagcctcagtttccccatctttaaaatgggagttatgttgagagattccagctgtggctcagcaggttaagaacccgactag
tatccatgaggaagagggttcaatccctggcttcgctcagcgggttaaggatccggcgttgccatgagctgcggcataag
tcgcagatgcagctcgaatcgggtgttgctgtggctgtggtgcaggctggcagctatcgcttccatcggacccctcgcctg
ggaacttccacgtatgccactggtgcagccctaaaagacaaacaaacaaaaacgaaagaaagagaaaagaaagg
aaagggggcttctgtttctaatgcgttgttgcctggcagggcgtgagcattagatacgtgtcagctgtgactagcgtgcacg
gagcacacaatccatgcttgtccagtaattagacaggctgggtgtccttccaccccctccctgcccaccagtgctctagag
aagcccacccaccagggctgggggagcacctgctctgtaccaggtaccgtgtgctgggagggggcagaggacctgat
ggctgtgaactggctcggtgcaggatgccggacagaggacgagccggggccgccgccggaggtgcacgtgtggtgg
gaggagcccaagtggcagggcagccagccctgggagcaggccctgggccgcttctgggattacctgcgctgggtgca
gtccctgtctgaccaagtgcaggaggagctgctcagcaccaaggtcacccaggaactgacgtaagtgcccacccgact
cccgccgcgcgcgcgcgcgcgcgcgcgcgcctgaccctcctggcggaccgtgtgttctggaccctcaggctccacccg
tccgggtttccttctgtccttgtcgccaactcttgggggtctgggtctctgtttcttttttttccttcttccttttttggggggagtttactttt
tcttttttctttcatttgacttcatgtcttgctttctttccatcttgagctcctgccttcgcctgtctctgggtcagtcttgccgtccttgctg
tctctgaatctctggcacgtcctggccatcgccagctcaggagccctccttctccccccccccgcccccgccctctctgcgc
ccagggagctgatagaggagagcatgaaggaggtgaaggcctaccgcgaggagctggaggcgcagctgggcccc
gtgacccaggagacgcaggcgcgcctgtccaaggagctgcaggcggcgcaggcccgcgtgggcgccgacatgga
ggacgtgcgcaaccgcttggtgctctaccgcagcgaggtgcacaacatgttgggccagaccaccgaggagctgcgga
gccgcctggcttcccacctgcgcaagctgcgcaagcggctgctccgcgacaccgaggacctgcagaagcgcctggcc
gtgtaccaggcggggctgcgcgagggcgccgagcgcagcgtgagcgccctccgcgagcgcctcgggcccctggtgg
agcagggccgattgcgcgccgccaccctgagtaccagggccggccagccgctgcgcgagcgcgccgaagcctggg
gccagaagctgcgcggacggctggaggagatgggcagccggacccgcgaccgcctggatgagatgcgtgagcagc
tggaggaggtgcgcaccaaagtggaggagcagggcagccagttgcgcctgcaggccgaggccttccaggcccgcct
caaaggctggttcgagcctctggtggaagacatgcggcgccagtgggccgggctggtggagaggatgcagtcggccg
tgagcatcagctcctccacctctgcgcccagtgataatcagtgagtgccctctcatccgggcacccccttcggggccccgt
tcctgcccaactcccccgcctcccccagccttagctgccctcttggtgggcccctgcttaataaagattcatcaagcttcaca
gcagcttctgggtgtc-3′

SEQ ID NO: 17: 6.1 kb ApoE fragment (3′-end of ApoE)—primers used for amplification are underlined:

[0236]

5′-
attcatcaagcttcacagcagcttctgggtgtccccgtgtgatttctcagctccagcctcagtttccctttccttccctgcactga
ccacccagttctctgtcctgccctctgcctgtgtgtgtctatttgtctcttctcccccttttctttttttttggccgagcccatggcatgc
ggaagttccccggccagggattgaacccatgccacagccgccacaacgaaggatccttaactactaggccaccaggg
aactccatcctttctaactctgtctttgctttcccttttttagcgttttagggctgcaccctcagcatgtggaagtccccaggctag
gggtcaaattggcgctacagctgccagcctacaccacagccccagcaacgcaggatccaagccacatctttgacctac
accacagctcatggtaacaccagatccttaacccactgagcaagggattgaacccacatcctcatggatactagtcggg
tttgttaatcactgagccacggcaggaaccccacccctgactactgtgggcaaaaaagcaacttcagagttcctgttgtgg
ctcagtgggttatgaacccaactagtatccatgagggtgcgggttcgatccctgatcctgctcagtgggttaaggatctgac
attgccatgagctccagtataggtaacagaaatgtcttgcatccacaccgctgtggctgtgacgtaggctggcagtttagct
ctgattcgacccctagcctgggaacttccttatgcccagggtttaaccctagaaaagagggggaaaaaaatcaacatctg
agcctcggttggcccagctttaaaatgcctgcttcatggccttgttactcaaaagacctgaaaccactgccatttggtttttttttt
taagtgtctttttttttttttaacgatttttattttttccattgtagttggtttacagcgttctgtgagttttctacggacccagtcacacac
atatatacattctttttgtcacatcatcctccatcctgctccatccccagtgactagatatagttcccagtgctctacagcaggat
ctcattgcttatcctctccagatgcaatggtttacgtctattaaacccagactcccagtccatcccacgccctccccttccccc
ccactgccatttttgttgagccattttcatttttttttcctccctctccctctcttacccgattctgcctcctttctgctcctggcctctgttc
tcagtcctgctctccctgagaggcttcatttctctggcttcctcttttcctccgcctctttctgtcctctccccctctggttgctcctgc
ccctggccctgcttgtttctagttgcccttcctccaggtttgccctcgccaccacgtgggccctctctttttttttttttttttacttcccc
cgaccaggaatcgaaccctagccatagaagtcacaatgccagatccttagctactagcccaccagggagttccatctcc
cctcatccttctctcctcccctggatcactggcctcttggctaccttgacaagcctaccaggtgctgggtgcaggctggaga
gaggggccagcctgtgacccttggtattaagggcggggccatcatgttgggagctgacacgcagcatggctggagcct
ggagaagcaggagcttccctcccacgccctcagttctcaggaggggagcaggattccatccagagccagcggacttgt
gtcttccaggcgggcctctgccccgcttggctctggtaaactctgtgctcactccgcgctttccctgccctgcttgccgctgtg
gaatcaggctccctccccccagccagatgttccacccttgggactgtgtgaggcggggctacatctgtgtgaggcagggc
caagtttctgctgattcactcactgtgtgtccagggcctgggcatctcattccccagatgtcggggagtggggctctcagcc
atatctcccattttaaaagctggatcttggagttcccttcatggctcgatggaagcaaatctggctagcatccatgaggatgc
gggttcgatccctggcctcactcagtaggttaaggatctggtgttgccgagagctggggtgtaggtagcagatggggctgt
ggctgtggtgcaggccggcagctgcagctccagtttgacccctaacctgggaacctccatgtgcactgggtgcggcccta
aaataaataaatgcatacgtaactaaatacatacatacatacatacataataaaaataaaaaaattaaaagctggatctc
aaattctgtttgaagccagctaggcggagaggggcgctcaccaccacaccccagcagcccaggttcctctctcagtgaa
aggaggctggcagggggggcagtggggtggcggctgaccccagcagggatccagagagtcagcctgaagggggg
aagatgatgaaggacagagaagggggcggcacgcagcctctcattgagcctctgaaccttcttagctgcccatcagtttc
cccctccctaaacggaggtgacagtgacgatgagactggccaaaccaagctgtcatccggggtggggaggggagga
gagcagacattcgggtggatgtggggagcgctgggctcacagaggaagcagccctcatcagaggggcctggggggc
tggcgggggctggatgcactcggagggctgttgcaatccggccagggtagcatctgtgcttgtctttcacaaccatcccct
cctcgccccaaggctgacacgtggttgttgggcacgaggccagccaacctagcgtctggggccagggcctctctccccc
agctgccagggatcacgagcagtcaaaggcagctggaggaagggggcagcctaggccggcagccctgccaacca
atgtggaggaagggacagggagagtgcgtggtggtaggagtggccaagagggggcatgagagcagatggagtgttt
ccagggacctggaggcttgcagaggcagggaacccagcgtcggggaacagggtttctggtggacccagtggagggc
acagattaggagccttgcagctgaggttctgcctctttttttattttagtgctgtacccgcggcatagggacgttctcagcctag
gagtcgaatcagagctgcagctgctggcctacaccacagccacgccagatccaagctgcttctgcgacctaaaccaca
gcttacagcaatgacggatccttgacccactgagtggggccagggatggaactggcatcctgagccacaacagaaact
catctgcacttctgacaggttcaggacaacctcctccaggagttccccattgtggcgcagcagaaacgaatccaactagg
aaccacgatgttgtaggttcgacccctggcctcgctcagtggcttaaggatctgacatgtgagctgtggtgtaggtcgcata
catggcttggatctagtgtttctgcggctgtggagtagagcagcagccgtagctcccatgggacccctaacctgggaacct
ccatgtgccgcaggtacggccctaaaaagaaaaaaaaaaaaaaaaaaaagagaaagagagagagaccctccact
gaaggaagattgggggctgtgaaattaaggctccagagagcgtccagggaggcctgggagtctcccagatgcagag
agaggggagaatggaagggctagtcggacagtgatattggagatggcatggtgggcaggtgtgtggaggcagactat
gagaccccagactcctgaagagtcttgagctgaagagacctactaagaaggggaggaggagttgccatcctggctca
gtggttaacgaatccgacgaggaaccatgaggttgcgggttcgatccccggcccctgctcagtgggttaaggatccggc
gttgccatgagctgtggtgtaggttgcagacgtggctcggatcccgtgtggctgtggctctggcgtaggccggcggctaca
gctccgattggacccctagcctgggaacctccatatgccatgggagcggcccaagaaatggcaaaaagacaaaaaa
aaaaagactccttccaagaacttgggtgctatgcactattaaggccatgaggggtaataccctcagagggcccagagat
gtaaagtcacacagccagcatgcggacaactggatcggggccccccagcctcaggcaatcactccactaccctcctcc
tgggctgggctgcccaagataaggaacattatcttgggctgattcaccaccaggcacacagaaggcatttattacacttctt
cttctttttttttttttttttaatttttgtcttttcagagctgcacccacagcttatagaggtacccaggctaggggtcgaatcagagc
agcagctgccagcctgcaccacagccacagcaacgtgggatccgagctgcatcttaaactaccccacagctcacagc
aacaccggatccttaacccactgagtgaggccagggatcgaacgaacctacgtcctcatggatgctactcaggttcattt
ccgctgagccacgatgggaactcctgttgattacactttcaaaggataatgaagggggatgtgagagaggtcaaaggtg
gacaagggctagagccctcaaacagaccgaccaacccccctctccaagtctcagctctgatgtcccctcctccaggaa
gccctccttgaccccaggttgaatcgagccccctcatctcagccctgtctactctgggtcatcactctctggggatggatgg
gcctgtccccccgaccccaccccaccccactggaccgtgagccctggggggacagggacagggcttcatcggcacc
atgctcaggcataacccagcacatgactaggcctggcacgggcactcattatttggtgaaacgagtatgctacctatgca
aagaaaataaataaacatgacattttcataaaaccctctgaggtagatttgtttccactgagccacgatgggagctccattt
aaaaatttttttaggagttcccgtcatggcgcagtggttaaccaatccgactaggaaccatgaggttgcgggttcgatccct
gcccttgctcagtgggttaacgatctggcgttgccgtgagctgtggtgcaggttgtagacgtggctcagttgctgtggccctg
gtgtgtaggccagcggcttcagctccgattagacccctagcctgggaacctccatgtgacgcaggagaggcccaagaa
atggctaggaatcatgaggttgcaggttcgatccctggccttgctcaatgggttaaggatccagtgttgtcgtgagctgtggt
gtaggttgcagatgaggctcagatcccacattgctgtggctctggcatgggctggcggctacagctccaattcgaccccta
gcctgggaacctccatatgccgtgggagcggctctagaaatggcaaaaagaccaaaagaaaaagaaaaaagaaa
aaaaaagaaaaagtgggcgggggccatagaggtggcctggggacacagtgtaaattgaattacttgtctggcttttttcttt
ctttctttttagggccgcaccggcggcttatggacatatggaggttcccaggctaggggtcgaatcggagctgcacctgggt
tctctcggggttccgctcaggctctctcaggctgcccccagggggtggtgatctgcccaggggagccctggcagccaatg
acgtagtcatgcccattcctccgggattggctgtcttgcttttacagctaagaaagggtggggtcctggtctagtgctgagag
gaaagcacgtcacagcctcttgagccccacctggtcgctctagtaccctctcctacattttaacaccatgacccccaagac
tcacattcaaggatctcctttaccatccctggagtctcaccccaagagctcccaatactgaatgttttgcacccctgcccctttt
ctgggtaggctcagccccagcctaggtgaccccag-3′

2. Transfection and Screening of Targeted Porcine Cells

[0237] Porcine fibroblasts from newborn or fetal Yucatan minipigs are cultured from ear biopsies. Cells are grown to 50% confluence in a 75 cm² flask (TPP, Switzerland), trypsinized and resuspended in 40 ml medium (DMEM, Lonza, Switzerland). One fourth (10 ml) were subsequently seeded in a 10 cm² petri dish. The cells are transfected with 6 ug of vector DNA in FuGENE 6 transfection reagent according to the protocol of the manufacturer (Roche Applied Science). The cells are grown under geneticin (Gibco Invitrogen) selection (1 mg/ml) for 8 days, neomycin resistant cell colonies were isolated, and each colony was transferred to a 1.9 cm² well (24-well plate, TPP, Switzerland) and grown to 80% confluence. Each colony is transferred to 9.4 cm² well (6-well plate TPP, Switzerland), grown to 80% confluence, and ⅓ of the cells is used for RNA isolation whereas ⅔ of the cells is transferred to a 25 cm² flask, grown to 80% confluence and stored at −135° C. in DMEM containing 10% DMSO until further use in handmade cloning.

[0238] Screening for locus-specific targeting events is performed by PCR and Southern Blotting using genomic DNA extracted from the neomycin-resistant clones as template. For PCR, a forward primer situated 5′-upstream for the ApoE homology region and a reverse primer located within the neomycin resistance gene, or a forward primer situated within the neomycin resistance gene and a reverse primer located 3′-downstream for the ApoE homology arm, is used.

[0239] Southern blot is performed to verify the locus-specific targeting events. Genomic DNA was digested and the blots probed with a DNA-probe situated outside the region of ApoE homology.

[0240] The resulting transgenic ApoE knockout porcine fibroblasts are subsequently used for somatic cell nuclear transfer (SCNT) by handmade cloning.

1. Cloning of Targeting Constructs

[0241] Part of ApoE intron 2 (left homology arm) and intron 3 (right homology arm) was amplified by PCR and used together with a gel extracted fragment of the vector pNeDaKO-Neo (containing a neomycin resistance gene) in a 3-fusion PCR as described by Kohli et al. (Nucleic Acids Research 2004, vol. 32, no. 1).

[0242] The resulting PCR product comprising the ApoE gene sequences flanking exon 3 and a neomycin resistance gene was cloned into the adeno-associated virus (AAV) vector pAAV-MCS (Stratagene). Packaging of this targeting construct (see below) and subsequent production of viral lysate for infection of porcine cells were performed as described by Kohli et al. (2004).

SEQ ID NO: 18: AAV-MCS/ApoE KO targeting construct—sequence (4054 bp)

(shaded region=Neo-sequence from pNeDaKO-Neo, unshaded regions=ApoE gene sequences, primer sequences are underlined)

[0243]

atacatacgcggccgcggatctgctcgggaaatatctgcttggattaggctgatgcagataagggggtgcaaggcttg
gaaggctgtgactggacagggctgggctctgggtgggaggagcgagccccgccgctgttgagtgacaatttctccctcct
gcaggttggccaatcgcaagccagaagatgagggttctgtgggttgctttggtggtaaccctcctcgcaggtatgggggtg
gggcttgctcaggttccctgcccctcccccatccccggctgtacccggtgcccctccttcatccctgggtctcttctgctggtct
ctcttccccttgaggagaggcctagatgtgaggcctctctggcactccttgcttctgaacagctcgttttactctctgagcctca
gtttccccatctttaaaatgggagttatgttgagagattccagctgtggctcagcaggttaagaacccgactagtatccatga
ggaagagggttcaatccctggcttcgctcagcgggttaaggatccggcgttgccatgagctgcggcataagtcgcagat
gcagctcgaatcgggtgttgctgtggctgtggtgcaggctggcagctatcgcttccatcggacccctcgcctgggaacttcc
acgtatgccactggtgcagccctaaaagacaaacaaacaaaaacgaaagaaagagaaaagaaaggaaaggggg
cttctgtttctaatgcgttgttgcctggcagggcgtgagcattagatacgtgtcagctgtgactagcgtgcacggagcacac
aatccatgcttgtccagtaattagacaggctgggtgtccttccaccccctccctgcccaccagtgctctagagaagcccac
ccaccagggctgggggagcacctgctctgtaccaggtaccgtgtgctgctaaagggaacaaaagctggagctccac
cgcggataacttcgtatagcatacattatacgaagttatcgcgccctaccgggtaggggaggcgcttttcccaaggcagtc
tggagcatgcgctttagcagccccgctgggcacttggcgctacacaagtggcctctggcctcgcacacattccacatcca
ccggtaggcgccaaccggctccgttctttggtggccccttcgcgccaccttctactcctcccctagtcaggaagttcccccc
cgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgcagatggacagcac
cgctgagcaatggaagcgggtaggcctttggggcagcggccaatagcagctttggctccttcgctttctgggctcagagg
ctgggaaggggtgggtccgggggcgggctcaggggcgggctcaggggcggggcgggcgcccgaaggtcctccgg
aagcccggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgggcctttcgacctgca
gccaatatgggatcggccattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggcta
tgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtc
aagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgt
tccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggat
ctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccgg
ctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcaatca
ggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgac
ggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcg
actgtggccggctgggtgtggcggatcgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggc
gaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacga
gttcttctgaggggatcaattctctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccct
cccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctg
agtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggca
tgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctgggggctgcagcacgtgttgacaattaatc
atcggcatagtatatcggcatagtataatacgactcactataggagggccaccatggccaagttgaccagtgccgttccg
gtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggagg
acgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaaca
ccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccggga
cgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccgg
caactgcgtgcacttcgtggccgaggagcaggactgaataacttcgtatagcatacattatacgaagttatggtacccaat
tcgccctatagtgagtcgtattaccggaccgtgtgttctggaccctcaggctccacccgtccgggtttccttctgtccttgtcgcc
aactcttgggggtctgggtctctgtttcttttttttccttcttccttttttggggggagtttactttttcttttttctttcatttgacttcatg
tcttgctttctttccatcttgagctcctgccttcgcctgtctctgggtcagtcttgccgtccttgctgtctctgaatctctggcacgtcc
tggccatcgccagctcaggagccctccttctccccccccccgcccccgccctctctgcgcccagggagctgatagagga
gagcatgaaggaggtgaaggcctaccgcgaggagctggaggcgcagctgggccccgtgacccaggagacgcagg
cgcgcctgtccaaggagctgcaggcggcgcaggcccgcgtgggcgccgacatggaggacgtgcgcaaccgcttggt
gctctaccgcagcgaggtgcacaacatgttgggccagaccaccgaggagctgcggagccgcctggcttcccacctgc
gcaagctgcgcaagcggctgctccgcgacaccgaggacctgcagaagcgcctggccgtgtaccaggcggggctgcg
cgagggcgccgagcgcagcgtgagcgccctccgcgagcgcctcgggcccctggtggagcagggccgattgcgcgc
cgccaccctgagtaccagggccggccagccgctgcgcgagcgcgccgaagcctggggccagaagctgcgcggac
ggctggaggagatgggcagccggacccgcgaccgcctggatgagatgcgtgagcagctggaggaggtgcgcacca
aagtggaggagcagggcagccagttgcgcctgcaggccgagggcggccgcgtatgtat

2. Transduction and Screening of Targeted Porcine Cells

[0244] Transduction of porcine fibroblasts from newborn or fetal Yucatan minipigs was performed in 75 cm² flasks as described by Kohli et al. (2004). Briefly, cells were infected with the virus for 2-3 hours and subsequently allowed to grow for 48 hours. The cells were harvested by trypsinization 48 hours post transduction and seeded into 96-well plates with media containing geneticin to select for targeted cells and allowed to grow for 2-3 weeks.

[0245] Initial screening for locus-specific targeting events was performed by PCR using genomic DNA extracted from the neomycin-resistant clones as template. For this PCR, a forward primer situated outside the left ApoE homology arm and a reverse primer located within the neomycin resistance gene, or a forward primer situated within the neomycin resistance gene and a reverse primer located outside the right ApoE homology arm, was used.

[0246] Southern blot was performed to verify the locus-specific targeting events. Genomic DNA was digested and the blots probed with a DNA-probe upstream for the left ApoE homology arm or downstream for the right ApoE homology arm.

[0247] The resulting transgenic ApoE knockout porcine fibroblasts were subsequently used for somatic cell nuclear transfer (SCNT) by handmade cloning.

[0248] In most cases of atherosclerosis the genetic component is complex, but in some cases the inheritance of the disease is monogenic. These cases are mostly caused by mutations in genes coding for proteins involved in lipoprotein trafficking, and the most severe in humans are caused by mutations affecting LDL receptor-mediated lipoprotein uptake (recessive and autosomal dominant familial hypercholesterolemia). Recently, a gain-of-function mutation in the PCSK9 gene was described as the cause of autosomal dominant familial hypercholesterolemia (17) PCSK9 binds to the LDL receptor leading to its degradation (18). Therefore, gain-of-function mutations in humans and overexpression of PCSK9 transgenes in mice leads to functional LDL receptor deficiency (19).

1. Cloning of Constructs

[0249] Donor cells transgenic for human proprotein convertase subtilisn/kexin type 9 (PCSK9) were produced using the DNA transposon-based vector pSBT-HCR-hAAT-PCSK9-bpA. Briefly, human PCSK9 was amplified by PCR from a PCSK9 cDNA clone (OriGene Technologies, Rockville, USA) using Pfx polymerase (Invitrogen) and the following primers.

[0250]

SEQ ID NO: 19:	Fw1 5′-AAAGGCGCGCCACCATGGGCACCGTCAGCTCCAGG-3′
SEQ ID NO: 20:	Rv1 5′-AAAGGCCGGCCTCACTCACTTGTCATCGTCGTCCTTGTAGTCC
	TGGAGCTCCTGGGAGGCCTG-3′

[0251] The forward primer includes an Ascl restriction site, consensus Kozak sequence and the beginning of the coding sequence of the human PCSK9 gene. The reverse primer includes a C-terminal FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), a stop codon and an FseI restriction site.

[0252] The human hepatocyte control region (HCR) of the apolipoprotein E gene followed by the human α₁-antitrypsin (hAAT) promoter was amplified by PCR from the plasmid pBS-apoEHCR-hAATp-hFIXmg-bpA (Miao C H et al. Mol. Ther. 2000; 1(6):522-532) (Pfx polymerase, 25 cycles, Invitrogen) using primers Fw2 and Rv2.

[0253]

SEQ ID NO: 21:	Fw2: 5′-AAATTAATTAACTAGT CTGCA GGCTC AGAGG-3′
SEQ ID NO: 22:	Rv2: 5′-GGCCGGCC GTTTAAAC GGCGCGCC GCAGATTGTGAAAGT
	GGTCG-3′

[0254] The bovine growth hormone polyadenylation signal (bpA) was amplified by PCR from pBS-apoEHCR-hAATp-hFIXmg-bpA using primers Fw3 and Rv3 (25 cycles, Pfx polymerase).

[0255]

SEQ ID NO:	Fw3:
23	5′-GGCGCGCCGTTTAAACGGCCGGCCTCAGCCTCGA
	CTGTGCCTTC-3′
SEQ ID NO:	Rv3:
24:	5′-GCGCTTAATTAAAGCCCCAGCTGGTTCCATAG-3′

[0256] The Rv2 and Fw3 primers contain a complementary region with Ascl and FseI restriction enzyme sites, and the HCR-hAAT and bpA segments were joined by fusion PCR using the primers Fw2 and Rv3. The fused PCR product was cloned into the unique PacI site of the Sleeping Beauty transposon-based vector pSBT-PGK-puro. (Yant and Kay. Mol Cell Biol. 2003; 23(23):8505-18). The human PCSK9 cDNA was thereafter cloned into the unique Ascl and FseI restriction sites.

Vector Construct for Liver-Specific Overexpression of a Native Human Pcsk9 Transgene

[0257] A Sleeping Beauty transposon-based vector was created containing the human hepatocyte control region (HCR) of the apolipoprotein E gene,²³ human α₁-antitrypsin (hAAT) promoter, human PCSK9 cDNA with a consensus Kozak sequence and an C-terminal FLAG tag,¹⁸ and a bovine growth hormone polyadenylation signal (bpA) (pSBT-PCSK9). The construct includes a puromycin resistance gene under the control of the PGK promoter.

2. Transfection

[0258] Porcine fibroblasts from newborn or fetal Yucatan minipigs were cultured in DMEM (Lonza, Switzerland) containing 15% fetal calf serum (Lonza). Cells were seeded in a 10 cm Petri dish at 1%, 0.25% or 0.1% confluency on day 0. On day 1, pSBT-HCRapoE-hAAT-PCSK9-BpA was co-transfected with a plasmid expressing the hyperactive Sleeping Beauty transposase mutant HSB3 (pCMV-HSB3, Yant S R et al. Mol Cell Biol. 2004; 24(20):9239-47) or the inactive transposase mutant mSB (pCMV-mSB, Yant et al. Nat. Genet. 2000; 25(1):35-41). From day 3, the cells were selected in 0.6 microgram/ml puromycin (Sigma) for 14 days. Number of resistant clones were counted (FIG. 1) and HSB3-tagged colonies were then cultured in 15% FCS in DMEM until hand-made cloning.

[0259] To confirm the integrity of the pSBT-HCRapoE-hAAT-PCSK9-BpA construct it was transiently transfected into the hepatocarcinoma cell line, HepG2. After 72 hours, the cells were stained for the PCSK9-FLAG protein using a biotinylated anti-FLAG antibody (1:1000, Sigma) and an Alexa 594-conjugated streptavidin (1:400) (FIG. 2).

3. Transgene Expression and Copy Number

[0260] Blood samples are obtained from newborn cloned pigs, and the presence and level of the transgenic PCSK9-FLAG protein in plasma is measured by ELISA using an anti-FLAG antibody (Sigma). Genomic DNAs of blood samples of the transgenic piglets and the surrogate mother are extracted according to the Chemagen DNA-extractor protocol. Southern blotting using a puromycin resistance gene (pac) probe is used to determine the number of integration sites.

[0261] Part of the pig LDL receptor coding sequence has been sequenced (GenBank accession no. AF065990). A targeting vector containing part of the gene sequence is created with a promoter-less neomycin resistance gene cassette inserted into one of the exons to disrupt gene function. The targeting vector is linearized and transfected into Yucatan fetal fibroblasts. Fibroblasts are cultured in the presence of G418. Resistant clones are screened for homologous recombination by PCR. Yucatan minipigs with knockout of one or both apoE alleles are created from recombinant cells by “hand-made” cloning.

[0262] Part of the pig LDL receptor coding sequence has been sequenced (GenBank accession no. AF065990). Efficient targets for RNA interference will be determined empirically by transient expression of vectors containing 1) a short hairpin RNA sequence expressed under a U6 or H1 promoter targeting a 19 nucleotide sequence in LDL receptor mRNA.

[0263] For stable short hairpin RNA expression, a transposon-based or retroviral vector is constructed with 1) a short hairpin RNA sequence expressed under an U6 or H1 promoter targeting a 19 nucleotide sequence in LDL receptor mRNA, 2) a puromycin resistance gene cassette, and 3) two flanking insulator sequences. The transposon-based targeting vector is cotransfected with transposase-expressing plasmid into Yucatan fetal fibroblast cultures. The retroviral vector is transduced into Yucatan fetal fibroblast cultures. Fibroblasts are cultured in the presence of puromycin. Resistant clones are screened for efficient down-regulation of LDL receptor mRNA and protein. LDL receptor knockdown Yucatan minipigs are created from transgenic cells by “hand-made” cloning.

Identification of Effective shRNAs

[0264] In this example, a human-sized model of atherosclerosis is created by development of genetically engineered Yucatan minipigs in which RNA effector molecules directed against the endogenous LDL-receptor induce reduce lipoprotein clearance and hypercholesterolemia.

[0265] Ten shRNA-expressing plasmids targeting different 19-nucleotide sequences in Yucatan minipig LDL receptor mRNA and one control pSUPER.retro.puro expressing an irrelevant shRNA was created in pSUPER.retro.puro (Oligoengine) using manufacturer's recommendations.

[0266]

TABLE 2
Targets for shRNA directed LDL receptor knockdown
	First base
	in pig cDNA
	AF065990
	sequence	Sequence
T1	763	tgtcaaagcggcgagtgca (SEQ ID NO: 5)
T2	889	tcccatatctgcaatgacc (SEQ ID NO: 6)
T3	1150	accctggaccgtagtgagt (SEQ ID NO: 7)
T4	1308	tgacaccattattggcgaa (SEQ ID NO: 8)
T5	1309	gacaccattattggcgaag (SEQ ID NO: 9)
T6	1439	agactctcttccaagagaa (SEQ ID NO: 10)
T7	1553	tgaacggagtggacgtcta (SEQ ID NO: 11)
T8	1814	tcacaggctcggacataca (SEQ ID NO: 12)
T9	858	ccaacgagtgtctggacaa (SEQ ID NO: 13)
T10	1109	cctacctcttcttcaccaa (SEQ ID NO: 14)

[0267] To measure the capacity of these shRNAs to target and facilitate degradation of LDL-receptor RNA we set up a Renilla luciferase screening-assay based upon targeting of a Renilla luciferase-LDL-receptor fusion mRNAs in HEK293 cells. Part of the coding sequence of the porcine LDL receptor was amplified by PCR from DNA obtained from Yucatan minipig fibroblasts using the following primers.

[0268]

SEQ ID NO: 25:	Fw
	5′ AAAACTAGTGCCAAGACGGGAAATGCATC 3′
SEQ ID NO: 26:	Rv
	5′ AAAACGGGTGCTGTTGATGCTCTTAAGCC 3′

[0269] The forward primer contained a SpeI site and the reverse an AgeI site and the LDL receptor segment was cloned into unique SpeI and AgeI sites in the 5′UTR region of the Renilla luciferase gene in a modified version of the pSiCheck-2 vector (Promega) to make the pSiCheck2-LDLR vector.

[0270] HEK293 cells were seeded at 19000 cells per well in 24-well plates (TPP) in DMEM containing 10% fetal calf serum (Lonza) on day 0. On day 1, cells were co-transfected with 0.04 μg pSiCheck2-LDLR and 0.36 μg pSUPER.retro.puro.T1-T10 or pSUPER.retro.puro.shScrambled using FuGene 6 transfection reagent (Roche Applied Science). On day 3, luciferase activity was measured using the Dual-Luciferase® Reporter Assay System from Promega. The assay was performed in triplicates. The experiment was repeated twice. Results are given in FIG. 4.

[0271] To facilitate stable expression of effective shRNAs in Yucatan minipig fibroblasts, shRNA sequences are cloned into one or both linkers of the plasmid pSBT/cHS4.H1p.PGK-puro.U6p.cHS4, in which shRNAs are expressed under an U6 or H1 polymerase III promoter, see FIG. 3.

Transfection

[0272] Porcine fibroblasts from newborn or fetal Yucatan minipigs are cultured in DMEM (Lonza, Switzerland) containing 15% fetal calf serum (Lonza). Cells are seeded in a 10 cm Petri dish at 1%, 0.25% or 0.1% confluency on day 0. On day 1, pSBT-shLDLR plasmid is co-transfected with a plasmid expressing the hyperactive Sleeping Beauty transposase mutant HSB3 (pCMV-HSB3, Yant S R et al. Mol Cell Biol. 2004; 24(20):9239-47). From day 3, the cells are selected in 0.6 microgram/ml puromycin (Sigma) for 14 days. Number of resistant clones are counted (FIG. 4) and HSB3-tagged colonies are then cultured in 15% FCS in DMEM until hand-made cloning.

Transgene Expression and Copy Number

[0273] Fibroblasts are cultured from newborn cloned pigs in DMEM supplemented with 10% fetal calf serum. For analysis of the level of LDL receptor knockdown, fibroblasts are cultured in DMEM supplemented with 10% lipoprotein-deficient serum for 48 hours and the level of LDL receptor mRNA is determined by quantitative PCR. Genomic DNA is isolated from fibroblasts by standard procedures. Southern blotting using a puromycin resistance gene (pac) probe is used to determine the number of integration sites.

[0274] Handmade cloning (HMC) and establishment of pregnancies for examples 1, 2, 3, 4 and 5.

[0275] For the cloning and delivery of transgenic fibroblasts are used in HMC. Recipient sows receive a total of in the range of 60-70 of a mixture of day 5 and/or 6 blastocysts.

[0276] Except where otherwise indicated all chemicals were obtained from Sigma Chemical Co. (St Louis, Mo., USA).

Oocyte Collection and In Vitro Maturation (IVM)

[0277] Cumulus-oocyte complexes (COCs) are aspirated from 2 to 6 mm follicles from slaughterhouse-derived sow ovaries and matured in groups of 50 in 400 μl IVM medium consisting of bicarbonate-buffered TCM-199 (GIBCO BRL) supplemented with 10% (v/v) cattle serum (CS), 10% (v/v) pig follicular fluid, 10 IU/ml eCG, 5 IU/ml hCG (Suigonan Vet; Skovlunde, Denmark) at 38.5° C. in 5% CO₂ in humidified air in the Submarine Incubation System (SIS; Vajta et al., 1997) for 41-44 h. HMC is performed by a procedure based on partial digestion of the zona pellucida, as described earlier (Du et al., 2005 and 2007). Matured COCs are freed from cumulum cells in 1 mg/ml hyaluronidase in Hepes-buffered TCM-199. From this point (except where otherwise indicated) all manipulations are performed on a heated stage adjusted to 39° C., and all drops used for handling oocytes are of 20 μl covered with mineral oil. Zonae pellucidae of are partially digested with 3.3 mg/ml pronase solution dissolved in T33 (T for Hepes-buffered TCM 199 medium; the number means percentage (v:v) of CS supplement, here 33%) for 20 s, then oocytes are washed quickly in T2 and T20 drops. Oocytes with distended and softened zonae pellucidae are lined up in T20 drops supplemented with 2.5 μg/ml cytochalasin B. With a finely drawn glass pipette, oocytes are rotated to locate the polar body on the surface. By oriented bisection with an Ultra Sharp Splitting Blade (AB Technology, Pullman, Wash., USA) less than half of the cytoplasm close to the polar body is removed manually from the remaining putative cytoplast.

[0278] Transgenic donor fibroblasts grown to a confluent monolayer in DMEM supplemented with 10% FCS were trypsinized and kept in T20 (Kragh et al., 2004). Fusion is performed in two steps. For the first step, 50% of the available cytoplasts are transferred into 1 mg/ml of phytohemagglutinin (PHA; ICN Pharmaceuticals, Australia) dissolved in TO for 3 s, then each one was quickly dropped over a single transgenic fibroblast. After attachment, cytoplast-fibroblast cell pairs are equilibrated in fusion medium (0.3 M mannitol and 0.01% PVA) for 10 s and transferred to the fusion chamber (BTX microslide 0.5 mm fusion chamber, model 450; BTX, SanDiego, Calif., USA). Using an alternating current (AC) of 0.6 kV/cm and 700 kHz, pairs are aligned to the wire of a fusion chamber with the somatic cells farthest from the wire, then fused with a direct current of 2.0 kV/cm for 9 μs. After the electrical pulse, cell pairs are incubated in T10 drops to observe whether fusion has occurred.

[0279] Approximately 1 h after the first fusion, each pair is fused with another cytoplast and activated simultaneously in activation medium (0.3 M mannitol, 0.1 mM MgSO₄, 0.1 mM CaCl₂ and 0.01% PVA). By using an AC of 0.6 kV/cm and 700 kHz, one fused pair and one cytoplast was aligned to one wire of the fusion chamber, with fused pairs contacting the wire, followed by a single DC pulse of 0.85 kV/cm for 80 μs. When fusion has been observed in T10 drops, reconstructed embryos are transferred into porcine zygote medium 3 (PZM-3; Yoshioka et al., 2002) supplemented with 5 μg/ml cytochalasin B and 10 μg/ml cycloheximide. After a 4 h incubation at 38.5° C. in 5% CO₂, 5% O₂ and 90% N₂ with maximum humidity, embryos are washed three times in PZM-3 medium before culture

Embryo Culture and Transfer

[0280] Embryos are cultured at 38.5° C. in 5% CO₂, 5% O₂ and 90% N₂ with maximum humidity in PZM-3 medium in the well of well system (WOWs; Vajta et al., 2000). Day 5 and 6 blastocysts with clearly visible inner cell mass are surgically transferred to Danish landrace sows on day 4 or 5 after weaning. Pregnancies are diagnosed by ultrasonography on day 21 and confirmed every second week. Piglets are delivered by Caesarean section on day 114, 24 h after treatment with prostaglandin F2.

[0281] On conventional diets (˜3% fat, w/w), Yucatan minipigs have low plasma cholesterol levels (˜2 mmol/I) and do not develop atherosclerosis. Transgenic minipig models of atherosclerosis has an expected total cholesterol level of >5 mM total cholesterol on normal pig diet, which can be further accentuated by feeding a diet containing 20% saturated fat and even more by feeding a diet containing 20% saturated fat and 1% cholesterol.

[0282] To characterize the level of hypercholesterolemia that can be obtained in transgenic Yucatan minipigs, transgenic and normal Yucatan minipigs are maintained on normal pig diet, pig diet supplemented with 20% saturated fat or pig diet supplemented with 20% saturated fat and 1% cholesterol. Blood samples are taken at time of weaning and every 3 months thereafter.

[0283] Plasma total cholesterol and triglycerides are measured on a Vitros 950 analyzer (Ortho-Clinical Diagnostics). HDL cholesterol (HDL-C) is measured enzymatically on a Kone 30 analyzer (Thermo) using kits from ABX (Triolab, Copenhagen, Denmark). The distribution of cholesterol within lipoprotein fractions is analyzed by fast protein liquid chromatography.

[0284] To characterize the level of atherosclerosis induced by the hypercholesterolemia, pigs are examined by intravascular ultrasound (IVUS) at 6 months, 9 months and 12 months of age. After the last examination, the minipigs are killed and the heart and all major arteries are removed and opened longitudinally to identify atherosclerotic lesions. Atherosclerotic lesions are fixed in 4% formaldehyde for 24 hours and embedded in paraffin. Specimens are serially sectioned, stained by elastin-trichrome and analyzed microscopically. Atherosclerotic lesions are characterized according to the classification described by Virmani et al. Transgenic pigs are expected to exhibit pathological intimal thickening and fibrous cap atheromas.

Molecular Imaging of Atherosclerosis:

[0285] Diagnostic imaging of atherosclerosis, which is becoming theoretically possible with the advent of new high-resolution imaging technology, is a promising new tool for risk stratification of asymptomatic persons. However, to develop tracers/contrast agents and imaging sequences that are able to visualize atherosclerotic plaques and atherosclerotic disease activity, we need a human-sized animal model of the disease that can be examined in patient CT, MR, and PET-scanners.

[0286] Example: Substance X is administered intravenously and accumulation in atherosclerotic plaques is analyzed by MR scanning, CT scanning, SPECT scanning, PET scanning or intravascular ultrasound. After the scanning, the minipigs are killed. Arterial specimens containing atherosclerotic lesions are fixed, paraffin-embedded, sectioned, stained and microscopically analyzed. Results obtained by imaging are compared to the histological findings.

[0287] Effect of stenting on atherosclerotic plaques: Plaques in the coronary arteries of a one-year old transgenic pig is mapped by intravascular ultrasound (IVUS) and a drug X-eluting stent and control stent is placed at the locations of two fibrous cap atheromas.

[0288] After 3 months, pigs are killed and the pathology of the stented atherosclerotic lesions is compared by histological techniques.

Drug Study:

[0289] Atorvastatin (3 mg/kg per day) or placebo is given to transgenic minipigs (6 months of age) for 3 months. Blood samples are obtained at the initiation of the study and every month hereafter. IVUS examinations of the coronary arteries are carried out at the initiation of the study and after 3 months. After the last IVUS examination, the minipigs are killed and atherosclerotic lesions are processed for histological analysis. The effect of atorvastatin on atherosclerosis is determined by comparing the serial IVUS data and the histology of atherosclerosis at study end between atorvastatin and placebo-treated minipig groups.

[0290] Based on the well-described mechanisms of SB transposition (4-8) and Flp recombination (9, 10), the present invention discloses a new target vector for site-specific integration into the genome. This vector carries within the context of a SB transposon vector a bicistronic gene cassette containing (i) the FRT recombination site embedded in the coding sequence of eGFP and (ii) an IRES-driven puromycin resistance gene. We demonstrate efficient selective plasmid insertion into SB-tagged genomic loci. In an attempt to further improve the performance of these vectors, we have analyzed the effect of insulator elements, believed to protect inserted foreign genes against transcriptional silencing, within the context of SB vectors. Our investigations indicate that insulators flanking the FRT gene expression cassette may serve to maintain and stabilize gene expression of Flp-inserted transgenes.

[0291] Two nonviral integration technologies are employed in the present invention, the SB transposon system and the Flp recombinase, in a combined effort to achieve active locus detection, mediated by SB, and site-directed insertion at an attractive site, mediated by Flp. A bi-phased technology is disclosed in which an integrating SB vector, carrying a reporter gene and a selective marker gene, may first serve as a reporter for continuous gene expression and hence as a target for gene insertion (FIG. 5). By using an actively integrated vector as opposed to plasmid DNA that is randomly recombined into the genome we certify (i) that only a single copy, and not concatemers, of the vector are inserted and, moreover, (ii) that the reporter cassette is not flanked by sequences derived from the bacterial plasmid backbone which may have a detrimental effect on the locus activity over time. In a second modification step this vector may serve as a target for insertion of one or more gene expression cassettes in a well-characterized locus.

Vector Construction

[0292] The SB transposon-based vector used in this study was derived from the pSBT/SV40-GFIP.loxP vector. This vector contains, within the context of a SB transposon, a bicistronic FRTeGFP-IRES-puro (GFIP) cassette flanked upstream by an ATG start codon and downstream by a poly A sequence. Moreover, the vector contains a recognition site for the Cre recombinase (loxP) located between the upper inverted repeat of the vector and the SV40 promoter driving expression of the FRTeGFP-IRES-puro cassette.

Construction of pSBT/SV40-GFIP.loxP Vector

[0293] The pSBT/RSV-GFIP vector contains the terminal inverted of the SB DNA transposon flanking a FRT-GFP.IRES.puro bicistronic gene cassette driven by a promotor derived from Rous sarcoma virus (RSV). The eGFP sequence was amplified from peGFP.N1 (Clontech) using a forward primer containing the 48-bp FRT sequence. To analyze FRT-GFP functionality, the FRT-eGFP fusion was inserted into an expression vector containing the SV40 promoter. The PCR-fragment containing FRT-tagged eGFP fusion gene was digested with Mlul and Xmal and inserted into Mlul/Xmal-digested pSBT/RSV-hAAT (pT/hAAT in ref. (8), obtained from Mark Kay, Stanford University, USA), generating a transposon vector with RSV-driven eGFP expression (pSBT/RSV-eGFP). An IRES-puro cassette was PCR-amplified from pecoenv-IRES-puro (provided by Finn Skou Pedersen, University of Aarhus, Denmark), digested with Xmal, and inserted into Xmal-digested pSBT/RSV-eGFP, generating pSBT/RSV-GFIP (see FIG. 6). Alternative versions of this vector containing the SV40 promoter (pSBT/SV40-GFIP) and the promoter derived from the human ubiquitin gene (pSBT/Ubi-GFIP), were generated. In addition, by inserting a Cre recombination target site (loxP) into the Mlul site located between the left inverted repeat of the transposon and the SV40 promoter of pSBT/SV40-GFIP, the vector pSBT/SV40-GFIP.loxP was created. The donor plasmid pcDNA5/FRT, containing a FRT-hygro fusion gene without a start codon, was obtained from Invitrogen. The Flp-encoding plasmid, pCMV-Flp was obtained from A. Francis Stewart, University of California San Francisco, USA). This plasmid encodes the enhanced Flp variant designated Flpx9 (11). A SB-vector containing two copies of the 1.2-kb chicken DNase hypersensitive site 4 (cHS4)-derived insulator element (12, 13) was generated by inserting PCR-amplified cHS4 sequences and an intervening linker into NotI/SpeI-digested pSBT/PGK-puro (obtained from Mark Kay, Stanford University, USA). The PGK-puro cassette was cloned back into construct by using restriction sites located in the linker, generating pSBT/cHS4.PGK-puro.cHS4

[0294] For further use in pigs an alternative Cre recognition site (loxP-257) was inserted into a unique Ascl site that was created by mutagenesis at a position located between the poly A sequence and the lower inverted repeat of the vector. This vector was designated pSBT/loxP.SV40-GFIP.loxP257. The presence of two Cre recombination sites allows Cre recombinase-mediated cassette exchange after Flp-based plasmid insertion, thereby facilitating, if needed, removal of plasmid sequences and selection genes.

SB Transposition in Primary Pig Fibroblasts

[0295] The SB transposon vectors, either SBT/PGK-puro or the target transposon SBT/loxP.RSV-GFIP.loxP257, were inserted into the genome of pig fibroblast by co-transfecting (using Fugene-6 from Roche) 1.5 μg pSBT/lox.RSV-GFIP.loxP257 (or pSBT/PGK-puro) with 1.5 μg pCMV-SB (or 1.5 μg pCMV-mSB as a negative control). pCMV-SB (rights held by Perry Hackett, University of Minnesota, Minnesota, USA) encodes the Sleeping Beauty transposase reconstructed from fossil DNA transposable elements of salmoid fish. pCMV-SB, pCMV-mSB, and the hyperactive variant pCMV-HSB3 were obtained from Mark Kay, Stanford University, USA. SB-tagged cell clones appeared as a result of selecting transfected cells with puromycin (0.5 μg/ml). Colonies were fixed and stained in methylene blue in methanol and subsequently counted.

Solid SB Transposition in Primary Pig Fibroblasts

[0296] SB transposes efficiently in most mammal cells but with higher efficacy in human cells than in murine cells. Transposition of SB vectors has never been analyzed in porcine cells, and we therefore initially tested activity in primary pig fibroblasts. We utilized a standard transposon encoding a puromycin resistance gene (SBT/PGK-puro) and found decent levels of transposition, resulting in about 75 drug-resistant colonies in cultures of fibroblasts co-transfected with pSBT/PGK-puro and pCMV-SB (FIG. 7). Less than 3 colonies appeared after transfection with pSBT/PGK-puro and pCMV-mSB, the latter which encodes an inactive version of the transposase. Interestingly, a mean of almost 140 colonies was obtained using the hyperactive transposase variant HSB3, indicating that HSB3 also in porcine cells mediates higher levels of transposition compared to the original SB transposase.

Efficient Insertion of a FRT-Tagged SB Vector in Pig Fibroblasts

[0297] To generate SB-tagged cell clones containing a Flp recombination target site for site-specific gene insertion, we co-transfected the pSBT/loxP.SV40-lopP257 plasmid with pCMV-mSB, pCMV-SB, and pCMV-HSB3, respectively. HSB3 again showed the highest activity, resulting in about 30 drug-resistant colonies after transfection of 3H 10⁴ fibroblasts (FIG. 8).

[0298] Puromycin-resistant colonies were isolated and expanded. Clone analysis by fluorescence microscopy demonstrated efficient FRTeGFP expression (FIG. 9), demonstrating vector functionality and easy FRTeGFP detection in pig fibroblasts. These fluorescent cell clones carrying the Flp FRT recombination sequence are currently being used for creation of cloned transgenic animals by hand-made cloning.

Verification of SBT/loxP.SV40-GFIP.loxP257 as Target for Flp Recombination

[0299] Due to limitations of long-term growth of primary pig fibroblasts in tissue culture we were not able to demonstrate Flp-based gene insertion into FRT-tagged SB vectors in pig fibroblasts. We therefore chose to test functionality of the FRT-containing vector by a standard set of recombination experiments carried out in HEK-293 cells. We generated clones of HEK-293 cells containing the transposed SBT/loxP.SV40-GFIP.loxP257 vector. By co-transfection of such clones with (i) a pcDNA5/FRT-derived substrate plasmid containing a FRT-hygro fusion gene and a red fluorescent protein (RFP) expression cassette and (ii) a plasmid encoding the Flp recombinase (pCMV-Flpx9), we subsequently identified hygromycin B resistant colonies. By fluorescence microscopy we observed that site-specifically engineered clones, as expected, turned-off eGFP expression and turned-on RFP expression (data not shown). This ‘green-to-red’ phenotypic change indicates that the integrated SB-derived target vector serves as acceptor site for Flp-based recombination.

[0300] In conclusion, the Sleeping Beauty DNA transposon-based vector of the present invention serves in its integrated form as a target for recombinase-based gene insertion. The SB vector is efficiently transferred by cut-and-paste transposition into the genome of primary porcine fibroblasts and therefore is not flanked by plasmid-derived bacterial sequences. Use of these genetically engineered primary cells in for example microinjection and hand-made cloning allows subsequent detailed analyses of SB vector-derived eGFP expression in cloned pigs and identification of animals with attractive expression profiles (e.g. ubiquitous, tissue-specific). Primary fibroblasts from such ‘master pigs’ is further modified by Flp-based recombination, allowing site-directed gene insertion in a SB vector-tagged locus which is not silenced in the tissue of interest. Cloned pigs harboring a site-specifically inserted disease gene of interest or a shRNA expression cassette for downregulation of endogenous genes can be generated by a second round of animal cloning.

[0301] Except where otherwise indicated all chemicals were obtained from Sigma Chemical Co. (St Louis, Mo., USA).

Oocyte Collection and In Vitro Maturation (IVM)

[0302] Cumulus-oocyte complexes (COCs) were aspirated from 2-6 mm follicles from slaughterhouse-derived sow or gilt ovaries. COCs were matured in groups of 50 in 400 μl bicarbonate-buffered TCM-199 (GIBCO BRL) supplemented with 10% (v/v) cattle serum (CS), 10% (v/v) pig follicular fluid, 10 IU/ml eCG, 5 IU/ml hCG (Suigonan Vet; Skovlunde, Denmark) at 38.5° C. in the “Submarine Incubation System” (SIS; Vajta, et al. 1997) in 5% CO₂ in humidified air for 41-44 hours.

In Vitro Fertilization (IVF)

[0303] IVF experiments were performed with in vitro matured oocytes in 3 identical replicates. After maturation, COCs were washed twice with mTBM containing 2 mM caffeine (mTBM_fert) and transferred in groups of 50 to 400 μl mTBM_fert. Freshly ejaculated semen was treated as described previously (Booth, et al., in press). After 2 h capacitation at 38.5° C. and in 5% CO₂ in humidified air, sperm was added to the oocytes with the adjusted final concentration of 1×10⁵ sperm/ml. Fertilization was performed at 38.5° C. and in 5% CO₂ in humidified air in the SIS for 3 h. After the insemination, the presumptive zygotes were vortexed in mTBM_fert to remove cumulus cells before washing in IVC medium and placing in culture dishes (see Embryo culture and evaluation).

Handmade Cloning (HMC)

[0304] The applied HMC method was based on our previous work in cattle and pig (Kragh, et al., 2004; Peura and Vajta, 2003; Vajta, et al., 2003), but with significant modifications. Briefly, at 41 h after the start of maturation, the cumulus investment of the COCs was removed by repeated pipetting in 1 mg/ml hyaluronidase in Hepes-buffered TCM199. From this point (except where otherwise indicated), all manipulations were performed on a heated stage adjusted to 39° C., and all drops used for handling oocytes were of 20 μl volume covered with mineral oil. Oocytes were briefly incubated in 3.3 mg/ml pronase dissolved in T33 (T for Hepes-buffered TCM 199 medium; the number means percentage (v/v) of CS supplement, here 33%) for 5 s. Before the oocytes started to become misshaped in pronase solution, they were picked out and washed quickly in T2 and T20 drops. Oocytes with partially digested but still visible zona were lined up in drops of T2 supplemented with 3 mg/ml polyvinyl alcohol (TPVA) and 2.5 μg/ml cytochalasin B. Trisection instead of bisection was performed manually under stereomicroscopic control with Ultra Sharp Splitting Blades (AB Technology, Pullman, Wash., USA; FIG. 10a). Fragments of trisected oocytes were collected and stained with 5 μg/ml Hoechst 33342 fluorochrome in TPVA drops for 5 min, then placed into 1 μl drops of the TPVA medium on the bottom of a 60 mm Falcon Petri dish covered with oil (3-4 fragments per drop). Using an inverted microscope and UV light, positions of fragments without chromatin staining (cytoplasts) were registered and later collected under a stereomicroscope in T10 drops until the start of the fusion.

[0305] Fetal fibroblast cells were prepared as described previously (Kragh, et al., in press). Fusion was performed in two steps where the second one included the initiation of activation, as well. For the first step, one third of the selected cytoplasts (preferably the smaller parts) were used. With a finely drawn and fire-polished glass pipette, 10 cytoplasts were transferred as a group to 1 mg/ml of phytohaemagglutinin (PHA; ICN Pharmaceuticals, Australia) for 3 s, then quickly dropped onto one of the few fibroblast cells individually that were sedimented in a T2 drop. After attachment, 10 cytoplast-fibroblast cell pairs were equilibrated in fusion medium (0.3 M mannitol and 0.01% PVA) for 10 s. Using an alternative current (AC) of 0.6 KV/cm and 700 KHz, cell pairs were aligned to the wire of a fusion chamber (BTX microslide 0.5 mm fusion chamber, model 450; BTX, SanDiego, Calif., USA) with the donor cells farthest from the wire (FIG. 10b), then fused with a direct current (DC) of 2.0 KV/cm for 9 μs. After the electrical pulse, cell pairs were removed carefully from the wire, transferred to T10 drops and incubated to observe whether fusion had occurred.

[0306] Approximately 1 hour after the first fusion, fused pairs together with the remaining two thirds of cytoplasts were equilibrated in activation medium drops separately (0.3 M mannitol, 0.1 mM MgSO₄, 0.1 mM CaCl₂ and 0.01% polyvinylalcohol (PVA)). Under a 0.6 KV/cm AC, cytoplast-fused pair-cytoplast triplets were aligned sequentially to the wire in groups of 10, with fused pairs located in the middle (FIG. 10c). A single DC pulse of 0.7 KV/cm for 80 μs was used for the second fusion and initiation of activation. The triplets were then removed from the wire and transferred carefully to T10 drops to check the fusion (FIG. 10d). Reconstructed embryos were incubated in culture medium (see Embryo culture and evaluation) supplemented with 5 μg/ml cytochalasin B and 10 μg/ml cycloheximide for 4 h at 38.5° C. in 5% CO₂, 5% O₂ and 90% N₂ with maximum humidity, then washed thoroughly for 3 times in IVC medium before culture.

Parthenogenetic Activation (PA)

[0307] Parthenogenetically activated oocytes were produced either separately or in parallel with HMC. Oocytes were denuded in the same way as above except that a longer incubation in pronase was used to get the zona pellucida completely removed. Zona free (ZF) oocytes were then equilibrated for 10 s in activation medium (0.3 M mannitol, 0.1 mM MgSO₄, 0.1 mM CaCl₂ and 0.01% PVA) and transferred to the fusion chamber (BTX microslide 0.5 mm fusion chamber, model 450; BTX, SanDiego, Calif., USA). A single DC pulse of 0.85 KV/cm for 80 μs was generated with a BLS CF-150/B cell fusion machine (BLS, Budapest, Hungary) and applied to ZF oocytes. For zona intact (ZI) oocytes, a single DC pulse of 1.25 KV/cm for 80 μs was used (according to our unpublished preliminary experiments, these parameters resulted in the highest activation and subsequent in vitro development for ZI and ZF oocytes, respectively). The procedure after the electrical pulse was the same as for HMC reconstructed embryos.

Embryo Culture and Evaluation

[0308] All porcine embryos produced by the above treatments were cultured in a modified NCSU37 medium (Kikuchi, et al., 2002) containing 4 mg/ml BSA at 38.5° C. in 5% O₂, 5% CO₂ and 90% N₂ with maximum humidity. The culture medium was supplied with 0.17 mm sodium pyruvate and 2.73 mm sodium lactate from Day 0 (the day for fertilization and activation) to Day 2, then sodium lactate and sodium pyruvate was replaced with 5.5 mm glucose from Day 2 to Day 7. All ZF embryos were cultured in the WOW system (Vajta, et al., 2000) in the same culture medium and gas mixture as used above, with careful medium change on Day 2 without removing the embryos from the WOWs. The blastocyst rate was registered on Day 7. To determine total cell numbers, blastocysts were fixed and mounted to a glass microscopic slide in glycerol containing 20 μg/μl Hoechst 33342 fluorochrome. After staining for 24 h, embryos were observed under a Diaphot 200 inverted microscope with epifluorescent attachment and UV-2A filter (Nikon, Tokyo, Japan).

10. 1

[0309] Differences in developmental competence between sow (2.5 years, 170 Kg in weight) derived oocytes and gilt (5.5˜6 months, 75 Kg in weight) derived oocytes were investigated through ZF and ZI PA after 44 h in vitro maturation. Four combined groups were investigated in 3 identical replicates: (1) ZF oocytes from sows (2) ZI oocytes from sows (3) ZF oocytes from gilts (4) ZI oocytes from gilts. For ZF activation, a single DC pulse of 0.85 KV/cm for 80 μs was applied, while a single 1.25 KV/cm pulse was used to activate ZI oocytes. Following 7 days culture as described above, the percentage of blastocysts per activated embryo was determined.

[0310] The in vitro developmental competence of parthenogenetically activated oocytes derived from either sows or gilts was investigated. As shown in Table 3, the blastocyst rates of parthenogenetically activated oocytes from sows were significantly higher than those from gilts, either after ZF or ZI PA.

[0311]

TABLE 3
Blastocyst development of Day 7 parthenogenetically
activated sow and gilt oocytes
	Zona Free		Zona Intact
	No. of	No. of	No. of	No. of
	activated	blastocysts	activated	blastocysts
	oocytes	(%)*	oocytes	(%)*
sow	103	43(42 ± 4)a	110	61(55 ± 6)c
gilt	85	17(20 ± 2)b	137	36(26 ± 5)d
a,bDifferent superscripts mean significant differences (p < 0.05).
c.dDifferent superscripts mean significant differences (p < 0.05).
*Percentage (Mean ± S.E.M) of embryos developed to blastocysts.

[0312] The difference in oocytes developmental competence between sows and gilts has been examined in in vitro production (IVP) and somatic cell nuclear transfer (SCNT) embryos separately, resulting in a similar conclusion as in the earlier publication of other research groups (Sherrer, et al., 2004; Hyun, et al., 2003), i.e. that embryos from sow-derived oocytes are superior to those from gilt-derived oocytes in supporting blastocyst development. Although gilts used in our study were at the borderline of maturity, the difference between Day 7 blastocyst rates after PA was significant, proving the superior developmental competence of sow oocytes.

10.2

[0313] The feasibility of modified porcine HMC was investigated in 6 identical replicates, with IVF and in parallel ZF PA as controls. The more competent sow oocytes (according to Example 1) were used in Example 2. Seven days after reconstruction and/or activation, the number of blastocysts per reconstructed embryo and total cell numbers of randomly selected blastocysts were determined.

[0314] More than 90% of oocyte fragments derived from morphologically intact oocytes could be recovered for HMC after the trisection. In average, 37 embryos could be reconstructed out of 100 matured oocytes. The developmental competence of all sources of porcine embryos is shown in Table 4. On Day 7, the development of reconstructed embryos to the blastocyst stage was 17±4% with mean cell number of 46±5, while the blastocyst rates for IVF, and ZF PA were 30±6% and 47±4% (n=243, 170, 97) respectively.

[0315]

TABLE 4
In vitro development of embryos
produced by HMC, IVF and ZF PA
	No. of		blastocyst	Mean cell
Embryo	embryos/oocytes	No. of	rates (Mean ±	number of
origins	in culture	blastocysts	S.E.M).	blastocysts
HMC	243	41	17 ± 4a	46 ± 5d
IVF	170	52	30 ± 6b	74 ± 6e
ZF PA	97	46	47 ± 4c	53 ± 7d
a,b,cDifferent superscripts mean significant differences (p < 0.05).
d,eDifferent superscripts mean significant differences (p < 0.05).

[0316] Although the theoretical maximum efficiency was still not approached, the integration of zona partial digestion and oocyte trisection almost doubled the number of reconstructed embryos compared to our earlier system (Kragh, et al., 2004 Reprod. Fertil. Dev 16, 315-318). This increase in reconstruction efficiency may have special benefits in porcine cloning since oocyte recovery after aspiration is more demanding and time-consuming than in cattle. An even more important point is the high embryo number required for establishment of pregnancies following porcine nuclear transfer. IVC in pigs is also regarded as a demanding and inefficient procedure (Reed, et al., 1992 Theriogeneology 37, 95-109). A disadvantage of ZF systems is that the embryos have to reach at least the compacted morula or early blastocyst stage in vitro to avoid disintegration in the oviduct without the protective layer of the zona pellucida. On the other hand, once in the blastocyst stage, zona free embryos can be transferred successfully as proved by calves born after either embryonic or somatic cell nuclear transfer (Peura et al., 1998; Tecirlioglu et al., 2004; Oback et al., 2003; Vajta, et al., 2004) and also by the piglets born after zona-free IVP of oocytes (Wu, et al., 2004). NCSU37 medium has been the most widely and successfully used medium for the culture of pig embryos. However, despite the improved embryo development compared with other media, the viability of IVP porcine embryos is still compromised after IVC. Some reports suggested that glucose is not metabolized readily by early porcine embryos before the eight-cell stage but used in higher amounts in embryos between the compacted morula and blastocysts stages (Flood, et al., 1988). The replacement of glucose with pyruvate and lactate in NCSU37 for the first 2 days culture resulted in a blastocyst rate of 25.3% for IVP porcine embryos in Kikuchi's study (Kukuchi, et al., 2002), which was further corroborated by our present studies with an IVP blastocysts rate of 30% in average. Moreover, the first evaluation of this sequential culture system on porcine HMC and ZF PA embryos has resulted in blastocyst rates of 17% and 47% respectively. Sometimes, the blastocyst rate of ZI PA could even reach levels as high as 90% (Du, unpublished)

Statistical Analysis

[0317] ANOVA analysis was performed using SPSS 11.0. A probability of P<0.05 was considered to be statistically significant.

10.3

[0318] Vitrification of hand-made cloned porcine blastocysts produced from delipated in vitro matured oocytes.

[0319] Recently a noninvasive procedure was published for delipation of porcine embryos with centrifugation but without subsequent micromanipulation (Esaki et al. 2004 Biol Reprod. 71, 432-6).

[0320] Cryopreservation of embryos/blastocysts was carried out by vitrification using Cryotop (Kitazato Supply Co, Fujinomiya Japan) as described previously (Kuwayama et al. 2005a; 2005b). At the time of vitrification, embryos/blastocysts were transferred into equilibration solution (ES) consisting of 7.5% (V/V) ethylene glycol (EG) and 7.5% dimethylsulfoxide (DMSO) in TCM199 supplemented with 20% synthetic serum substitute (SSS) at 39° C. for 5 to 15 min. After an initial shrinkage, embryos regained their original volume. 4˜6 embryos/blastocysts were transferred into 20 ul drop of vitrification solution (VS) consisting of 15% (V/V) EG and 15% (DMSO) and 0.5M sucrose dissolved in TCM199 supplemented with 20% SSS. After incubation for 20 s, embryos were loaded on Cryotop and plunged into liquid nitrogen. The process from exposure in VS to plunging was completed with 1 min.

[0321] Embryos/blastocysts were thawed by immersing Cryotop directly into thawing solution (TS) consisting of 1.0M sucrose in TCM199 plus 20% SSS for 1 min, then transferred to dilution solution (DS) consisting of 0.5 M sucrose in TCM199 plus 20% SSS for 3 min. To remove cryoprotectant, embryos/blastocysts were kept twice in a washing solution (WS; TCM199 plus 20% SSS), 5 min for each time. Survival of vitrified blastocysts was determined according to reexpansion rates after 24 h recovery in culture medium supplemented with 10% calf serum (CS).

[0322] The non-invasive delipation method was applied to in vitro matured porcine oocytes and further development of delipated oocytes after parthenogenetic activation was investigated in 4 identical replicates. Oocytes were randomly separated into delipation and control groups.

[0323] For delipation, oocytes were digested with 1 mg/ml pronase in the presence of 50% cattle serum (CS) for 3 min, and washed in Hepes-buffered TCM-199 medium supplemented with 20% CS which results in partial zona pellucida digestion (FIG. 11a). Subsequently 40-50 oocytes were centrifuged (12000×g, 20 min) at room temperature in Hepes-buffered TCM-199 medium supplemented with 2% CS, 3 mg/ml PVA and 7.5 μg/ml cytochalasin B (CB) (FIG. 11b). Zonae pellucidea of both centrifuged and intact oocytes were removed completely with further digestion in 2 mg/ml pronase solution. For activation, a single direct current of 85 Kv/cm for 80 us was applied to both groups, followed by 4 h treatment with 5 μg/ml CB and 10 μg/ml cycloheximide (CHX). All embryos were then cultured in the modified NCSU37 medium. Day 7 blastocysts were vitrified and warmed by using the Cryotop technique (Kuwayama et al., RBM Online, in press) at 38.5° C. Survival of vitrified blastocysts was determined according to reexpansion rates after 24 h recovery in culture medium supplemented with 10% CS. Cell numbers of reexpanded blastocysts from both groups were determined after Hoechst staining. Results were compared by ANOVA analysis. Partial zona digestion and centrifugation resulted in successful delipation in 173/192 (90%) of oocytes. The development to blastocysts was not different between delipated and intact oocytes (28±7% vs.28±5% respectively; P>0.05). However, survival rates of blastocysts derived from delipated oocytes were significantly higher than those developed from intact oocytes (85±6% vs.32±7% respectively; P<0.01). There is no difference in average cell number of reexpanded blastocysts derived from either delipated or intact oocytes (36±7 vs. 38±9, respectively; P>0.05). The results demonstrate that the simple delipation technique does not hamper the in vitro development competence of activated porcine oocytes, and improves the cryosurvival of the derived blastocysts without significant loss in cell number.

[0324] After delipation, zona pellucida of oocytes from both groups was removed completely. The same parameters as described above for electrical activation were applied to both groups. Seven days after activation, blastocyst rates and blastocyst cell numbers were determined.

[0325] The feasibility of applying a non-invasive delipation technique to in vitro matured porcine oocytes was investigated. 90% (173/192) oocytes can be delipated successfully. As shown in table 5, the development to blastocysts was not different between delipated and intact oocytes (28±7% vs.28±5% respectively; P>0.05). However, survival rates of blastocysts derived from delipated oocytes were significantly higher than those developed from intact oocytes (85±6% vs.32±7% respectively; P<0.01). There is no difference in average cell number of reexpanded blastocysts derived from either delipated or intact oocytes (36±7 vs. 38±9, respectively; P>0.05).

[0326]

TABLE 5
Developmental competence and cryosurvival of vitrified-thawed
embryos from delipated and intact activated oocytes.
			Reexpanded	Mean cell number
Oocyte	Activated	Blastocyst	blastocyst after	of reexpanded
treatment	oocyte	rate (%)	warming (%)	blastocysts
Delipated	173	28 ± 7	85 ± 6	36 ± 7
Intact	156	28 ± 5	32 ± 7	39 ± 9

Handmade Cloning of Delipated Oocytes

[0327] Delipated oocytes were used for HMC in 5 replicates. Four identical replicates of non-delipated oocytes for HMC were used as a control system. Seven days after reconstruction, blastocysts produced from both groups were vitrified with Cryotop. Survival rates and cell numbers of re-expanded blastocysts were determined as described for the blastocysts produced by PA.

[0328] Except where otherwise indicated, all manipulations were performed on a heated stage adjusted to 39° C., and all drops used for handling oocytes were of 20 μl volume covered with mineral oil. For somatic cell nuclear transfer, the handmade cloning (HMC) described in our previous work (Du, et al., 2005) was applied with a single modification: for enucleation of both delipated and control oocytes, bisection instead of trisection was applied.

[0329] Briefly, after the removal of cumulus investment, control oocytes were incubated in 3.3 mg/ml pronase dissolved in T33 for 10 s. Before the oocytes started to become misshaped in pronase solution, they were picked out and washed quickly in T2 and T20 drops. Delipated oocytes after centrifugation were digested in the 3.3 mg/ml pronase solution for an additional 5 s.

[0330] Both control and delipated oocytes with partially digested, distended and softened zonae pellucidae were lined up in T2 drops supplemented with 2.5 μg/ml cytochalasin B. Bisection was performed manually under stereomicroscopic control (FIG. 11c) with Ultra Sharp Splitting Blades (AB Technology, Pullman, Wash., USA). Halves were collected and stained with 5 μg/ml Hoechst 33342 fluorochrome in T2 drops for 5 min, and then placed into 1 μl drops of T2 medium on the bottom of a 60 mm Falcon Petri dish covered with oil (3-4 halves per drop). Using an inverted microscope and UV light, positions of halves without chromatin staining (cytoplasts) were registered. Cytoplasts were later collected under a stereomicroscope and stored in T10 drops.

[0331] Porcine foetal fibroblast cells were prepared with trypsin digestion from monolayers as described previously (Kragh, et al., 2005). Fusion was performed in two steps where the second one included the initiation of activation, as well. For the first step, 50% of the available cytoplasts were transferred into 1 mg/ml of phytohaemagglutinin (PHA; ICN Pharmaceuticals, Australia) dissolved in TO for 3 s, then quickly dropped over single fibroblast cells. After attachment, cytoplast-fibroblast cell pairs were equilibrated in fusion medium (0.3 M mannitol and 0.01% PVA) for 10 s and transferred to the fusion chamber. Using an alternating current (AC) of 0.6 KV/cm and 700 KHz, pairs were aligned to the wire of a fusion chamber with the somatic cells farthest from the wire (FIG. 11d), then fused with a direct current of 2.0 KV/cm for 9 μs. After the electrical pulse, cell pairs were removed carefully from the wire, transferred to T10 drops and incubated to observe whether fusion had occurred.

[0332] Approximately 1 hour after the first fusion, each pair was fused with another cytoplast in activation medium. AC current and a single DC pulse of 0.7 KV/cm for 80 μs were applied as described above. Fusion was detected in T10 drops, then reconstructed embryos were transferred into IVC0-2 medium (see Embryo culture and evaluation) supplemented with 5 μg/ml cytochalasin B and 10 μg/ml cycloheximide. After a 4 h incubation at 38.5° C. in 5% O₂, 5% O₂ and 90% N₂ with maximum humidity, embryos were washed 3 times in IVC0-2 medium before culture.

[0333]

TABLE 6
Developmental competence and cryosurvival of
vitrified-thawed embryos of SCNT porcine embryos
derived from delipated and intact oocytes.
				Mean cell
	No. of		Reexpanded	number of
HMC	reconstructed	Blastocyst	blastocyst after	reexpanded
group	embryos	rate (%)*	warming (%)*	blastocysts*
Delipated	240	21 ± 6a	79 ± 6b	41 ± 7d
Intact	150	23 ± 6a	32 ± 8c	39 ± 5d
Different superscripts mean significant differences (p < 0.05).
*mean ± S.E.M.

[0334] In vitro developmental competence was observed in HMC with delipated oocytes when Day 7 blastocyst rates were compared with control HMC group (21±6% vs.23±6% respectively; P>0.05; Table 6). Cryosurvival rate after vitrification of cloned blastocysts derived from delipated oocytes was significantly higher than those developed from intact oocytes (79±6% vs. 32±8, respectively; P<0.01).

10. 4

Chemically Assisted Handmade Enucleation (CAHE) and Comparison to Existing Methods

[0335] After 41-42 h maturation in vitro, COCs were further cultured for 45 min in the same solution supplemented by 0.4 μg/ml demecolcine. Cumulus cells were then removed by pipetting in 1 mg/ml hyaluronidase dissolved in Hepes-buffered TCM-199. From this point (except where otherwise indicated), all manipulations were performed on a heated stage adjusted to 39° C. All drops used for handling oocytes were of 20 μl in volume, and were covered with mineral oil.

[0336] Basic steps of the HMC procedure have been described elsewhere herein. Briefly, oocytes without cumulus cells were incubated in 3.3 mg/ml pronase dissolved in T33 (T for Hepes-buffered TCM 199 medium; the number means percentage [v/v] of CS supplement, here 33%) for 20 s. When partial lyses of zonae pellucidae and slight deformation of oocytes occurred, they were picked up and washed quickly in T2 and T20 drops. Nine oocytes were lined up in one T2 drop supplemented with 2.5 μg/ml cytochalasin B (CB). By using a finely drawn and fire-polished glass pipette, oocytes were rotated to find a light extrusion cone and/or strongly attached polar body on the surface, and oriented bisection was performed manually under stereomicroscopic control with a microblade (AB Technology, Pullman, Wash., USA). Less than half of the cytoplasm (close to the extrusion or PB) was separated from the remaining part (FIG. 12). After bisection of all 9 oocytes in the drop, larger parts and smaller parts (with the extrusion or attached PB) were collected and placed into separate drops of T2, respectively.

Oriented Handmade Enucleation without Demecolcine Treatment (OHE)

[0337] All steps were similar to the previously described procedure, but demecolcine preincubation was not applied.

Random Handmade Bisection for Enucleation (RHE)

[0338] Demecolcine preincubation was omitted from the pretreatment of this group, as well. After removal of cumulus cells, zonae pellucidae were partially digested by pronase as described above. Random handmade equal bisection was applied in drops of T2 supplemented with 2.5 μg/ml CB. All demi-oocytes were selected and stained with 10 μg/ml Hoechst 33342 in T2 drops for 10 min, then placed into 1 μl drops of T2 medium covered with mineral oil (three demi-oocytes into each drop). Using an inverted microscope and UV light, the positions of chromatin free demi-oocytes, i.e. cytoplasts were registered. These cytoplasts were later collected under a stereomicroscope and stored in T2 drops before further manipulations.

Fusion and Initiation of Activation

[0339] Porcine fetal fibroblast cells were prepared as described previously (Kragh, et al., 2005, Du, et al., 2005). Fusion was performed in two steps, where the second one included the initiation of activation as well. For the first step, with a finely drawn and fire-polished glass pipette, approximately 100 somatic cells were placed into a T2 drop, and 20-30 cytoplasts were placed into a T10 drop. After a short equilibration, groups of 3 cytoplasts were transferred to 1 mg/ml of phytohaemagglutinin (PHA) for 2-3 sec, then each was quickly dropped over a single somatic cell. Following attachment, cytoplast-somatic cell pairs were picked up again and transferred to a fusion medium (0.3 M mannitol supplemented with 0.01% [w/v] PVA). By using an alternative current (AC) of 0.6 KV/cm and 700 KHz, equilibrated pairs were aligned to one wire of a fusion chamber (BTX microslide 0.5 mm fusion chamber, model 450; BTX, San Diego, Calif.) with the somatic cells farthest from the wire, then fused with a single direct current (DC) impulse of 2.0 KV/cm for 9 μsec. Pairs were then removed carefully from the wire to a T10 drop, and incubated further to observe whether fusion had occurred.

[0340] Approximately 1 h after the fusion, fused pairs and the remaining cytoplasts were separately equilibrated in activation medium (0.3 M mannitol, 0.1 mM MgSO₄, 0.1 mM CaCl₂, supplemented with 0.01% [w/v] PVA). By using a 0.6 KV/cm AC, one pair and one cytoplast was aligned to one wire of the fusion chamber, with fused pairs contacting the wire. A single DC pulse of 0.86 KV/cm for 80 μsec was used for the second fusion and initiation of activation. Fusion was checked in after incubation in T10 drops.

Traditional Cloning (TC)

[0341] Micromanipulation was conducted with a Diaphot 200 inverted microscope (Nikon, Tokyo, Japan), as described before (Chen et al., 1999; Zhang et al., 2005). Briefly, after 42-44 h in vitro maturation, the cumulus cells were removed as described above. All manipulations were performed on a heated stage adjusted to 39° C. A single 50 μL micromanipulation solution drop was made in the central area on a lid of 60 mm culture dish and covered with mineral oil. Groups of 20-30 oocytes and fetal fibroblast cells were placed in the same drop. After incubation for 15-30 min, the oocyte was secured with a holding pipette (inner diameter=25-35 μm and outer diameter=80-100 μm). After being placed at the position of 5-6 o'clock, the first polar body and the adjacent cytoplasm (approx. 10% of the total volume of the oocyte) presumptively containing metaphase plate were aspirated and removed with a beveled injection pipette (inner diameter=20 μm). A fetal fibroblast cell was then injected into the space through the same slit. After nuclear transfer (NT), reconstructed couplets were transferred into drops of media covered with mineral oil for recovery for 1-1.5 h until fusion and activation was conducted. The recovery medium was NCSU-23 supplemented with 4 mg/mL BSA and 7.5 μg/mL CB. Reconstructed couplets were incubated in fusion medium for 4 min. Couplets were aligned manually using a finely pulled and polished glass capillary to make the contact plane parallel to electrodes. A single, 30 μsec, direct current pulse of 2.0 kV/cm was then applied. After culture in drops of IVC0-2 (specified in “Embryo culture and evaluation”) supplemented with 7.5 μg/mL CB for 30-60 min, fusion results were examined under a stereomicroscope. Fused couplets were subjected to a second pulse in activation solution. After 30 min incubation in T10 they were transferred to IVC0-2 to evaluate in vitro development.

Further Steps of Activation

[0342] After the activation impulse, all reconstructed embryos were incubated in IVC0-2 supplemented with 5 μg/ml CB and 10 μg/ml cycloheximide at 38.5° C. in 5% CO₂, 5% O₂, and 90% N₂, with maximum humidity.

Embryo Culture and Evaluation

[0343] 4 h later, all reconstructed and activated embryos were washed and cultured in Nunc four-well dishes in 400 μl IVC0-2 covered by mineral oil at 38.5° C. in 5% CO₂, 5% O₂, and 90% N₂, with maximum humidity. IVC0-2 was a modified NCSU37 medium (Kikuchi, et al., 1999), containing 4 mg/ml BSA, 0.17 mM sodium pyruvate, and 2.73 mM sodium lactate from Day 0 (the day for activation) to Day 2. Sodium pyruvate and sodium lactate were replaced with 5.5 mM glucose from Day 2 to Day 7 (IVC2-7). All zonae free embryos were cultured in the Well of the Well (WOW) system (Vajta et al., 2000) in the same culture medium and gas mixture as used above, with careful medium change on Day 2 without removing the embryos from the WOWs. TC embryos were cultured in groups of 15 to 30 in wells of four-well dishes by using the same medium amount and composition. Cleavage and blastocyst rates were registered on Day 2 and Day 7, respectively. To determine total cell numbers, blastocysts were fixed and mounted to a glass microscope slide in a small amount (<2 μl) of glycerol containing 10 μg/ml Hoechst 33342. After staining for several hours at room temperature, embryos were observed under a Diaphot 200 inverted microscope with epifluorescent attachment and UV-2A filter (Nikon, Tokyo, Japan).

Comparison of Efficiency of CAHE vs. OHE

[0344] The efficiency and reliability of CAHE was tested in 12 identical replicates by using a total of 620 oocytes. After 41-42 h maturation, oocytes were subjected to demecolcine incubation. Oriented bisection was performed in oocytes where an extrusion cone and/or a strongly attached PB was detected after partial pronase digestion. Percentages of bisected vs. total oocytes and surviving vs. bisected oocytes were registered. Subsequently both putative cytoplasts and karyoplasts were collected separately and stained with Hoechst 33342 (10 μg/ml in T2 for 10 min). The presence or absence of chromatin was detected under an inverted fluorescent microscope (FIG. 13).

[0345] The efficiency and reliability of OHE was investigated in 9 identical replicates using a total of 414 oocytes. After 42-43 h in vitro maturation, oriented bisection was performed in matured oocytes where an extrusion cone and/or a PB was detected after partial pronase digestion. Results were evaluated as described in the previous paragraph.

[0346] The results are shown in Table 7.

[0347]

TABLE 7
The efficiency of chemically assisted handmade enucleation
(CAHE) and oriented handmade enucleation (OHE)
	No. of	Bisected/		Cytoplast/
	treated	total	Cytoplast/	total
Groups	oocytes	oocytes (%)*	bisection (%)*	oocyte (%)*
CAHE	620	96 ± 1a	94 ± 2b	90 ± 3c
OHE	414	92 ± 2a	88 ± 3b	81 ± 4d
*mean ± A.D. (absolute deviations)
Different superscripts mean difference (P < 0.05)

[0348] No differences between groups regarding extrusion cones and/or attached polar bodies allowing oriented bisection or in the lysis rates were detected, and the successful enucleation per bisected oocyte ratio was also similar. However the overall efficiency of the procedure measured by the cytoplast per total oocyte number was higher in the CAHE than in the OHE group.

Comparison of In Vitro Development of Embryos Produced with CAHE, RHE and TC

[0349] In 8 replicates, a total of 468 in vitro matured oocytes were randomly distributed and subjected to three of the enucleation procedures described above. Fusion rates between cytoplast and donor fibroblasts were registered. Reconstructed embryos were activated and cultured as described earlier. Cleavage and blastocyst rates were determined on Day 2 and Day 7, respectively. Stereomicroscopic characteristics of the developed blastocysts were compared between groups.

[0350]

TABLE 8
Developmental competence of embryos derived from chemically assisted
handmade enucleation (CAHE), random handmade enucleation (RHE)
and traditional, micromanipulator based cloning (TC).
	No. of				Cell no. of
	reconstructed	Fusion	Cleavage	Blastocyst	blastocysts
Groups	embryos	rate (%)*	rate (%)*	rate (%)*	(Day 7)
CAHE	150	87 ± 7a	97 ± 6b	28 ± 9d	57 ± 6e
RHE	86	81 ± 4a	87 ± 8b	21 ± 9d	49 ± 7e
TC	178	81 ± 10a	69 ± 9c	21 ± 6d	53 ± 6e
*mean ± A.D. (absolute deviations)
Different superscripts mean difference (P < 0.05).

[0351] Fusion rates after enucleation were similar between CAHE, RHE and TC, respectively. The second fusion and activation resulted in negligible (<1%) losses in the first two groups. Although TC resulted in lower cleavage per reconstructed embryo rates than the other two groups, this difference was not present in the blastocyst per reconstructed embryo rates.

[0352] Stereomicroscopic characteristics (size; estimated proportion and outlines of the inner cell mass) did not differ between groups. Cell numbers (57±6 vs. 49±7 vs. 53±6) of the produced blastocysts from CAHE, RHE and TC are shown in Table 8, FIG. 14 and FIG. 15.

Statistical Analysis

[0353] AVEDEV was performed by Microsoft XP Excel software and ANOVA was performed by SAS system. A probability of P<0.05 was considered to be statistically significant.

10.5

Production of Piglets

Handmade Cloning (HMC)

[0354] Forty one hrs after the start of in vitro maturation, the cumulus investment of the COCs was removed by repeated pipetting in 1 mg/ml hyaluronidase in Hepes-buffered TCM199. From this point (except where otherwise indicated) all manipulations were performed on a heated stage adjusted to 39° C., and all drops used for handling oocytes were of 20 μl volume covered with mineral oil. Oocytes were briefly incubated in 3.3 mg/ml pronase dissolved in T33 (T for Hepes-buffered TCM 199 medium; the number means percentage (v/v) of calf serum (CS) supplement, here 33%) for 20 sec and then quickly washed in T2 and T20 drops. Oocytes with partially digested but still visible zona were lined up in drops of T2 supplemented with 2.5 μg/ml cytochalasin B (CB). With a finely drawn and fire-polished glass pipette, oocytes were rotated to find the polar body (PB) on the surface, and oriented bisection was performed manually under stereomicroscopic control with a microblade (AB Technology, Pullman, Wash., USA). Thus, less than half of the oocyte cytoplasm (close to the extrusion or PB) was removed from the remaining putative cytoplast. Cytoplasts were washed twice in T2 drops and collected in a T10 drop.

[0355] Fetal fibroblast cells were prepared as described previously (Kragh, P. M. et al. Theriogenology 64, 1536-1545 (2005).

[0356] Fusion was performed in two steps where the second one included the initiation of activation, as well. For the first step, halves of putative cytoplasts were used. With a finely drawn and fire-polished glass pipette, 10 cytoplasts were transferred as a group to 1 mg/ml of phytohaemagglutinin (PHA; ICN Pharmaceuticals, Australia) for 3 sec, then quickly dropped individually onto one of the few fibroblast cells that were sedimented in a T2 drop. After attachment, 10 cytoplast-fibroblast cell pairs were equilibrated in fusion medium (0.3 M mannitol and 0.01% PVA) for 10 sec. Using an alternative current (AC) of 0.6 KV/cm and 700 KHz, cell pairs were aligned to the wire of a fusion chamber (BTX microslide 0.5 mm fusion chamber, model 450; BTX, SanDiego, Calif., USA) with the somatic cells farthest from the wire, then fused with a direct current (DC) of 2.0 KV/cm for 9 μsec. After the electrical pulse, cell pairs were removed carefully from the wire, transferred to T10 drops and incubated to observe whether fusion had occurred.

[0357] Approximately 1 hr after the first fusion, fused pairs together with the remaining cytoplasts were equilibrated in activation medium drops separately (0.3 M mannitol, 0.1 mM MgSO₄, 0.1 mM CaCl₂ and 0.01% PVA). Under a 0.6 KV/cm AC, cytoplast-fused pair were aligned sequentially to the wire in groups of 10, with fused pairs far from the wire. A single DC pulse of 0.7 KV/cm for 80 μsec was used for the second fusion and initiation of activation. The pairs were then removed from the wire and transferred carefully to T10 drops to check the fusion. Reconstructed embryos were incubated in PZM-3 medium supplemented with 5 μg/ml CB and 10 μg/ml cycloheximide for 4 hr at 38.5° C. in 5% O₂, 5% O₂ and 90% N₂ with maximum humidity, then washed thoroughly before culture.

Traditional Cloning (TC)

[0358] Micromanipulation was conducted with a Diaphot 200 inverted microscope (Nikon, Tokyo, Japan). Cumulus cells were removed as described above after 42 to 44 hr maturation. All manipulations were performed on a heated stage adjusted to 39□. A single 50 μL drop of micromanipulation solution (NCSU-23 supplemented with 4 mg/mL BSA and 7.5 μg/mL CB) was made in the central area on a lid of 60 mm culture dish and covered with mineral oil. Groups of 20 to 30 oocytes and fetal fibroblast cells were placed in the same drop. After incubation for 15 to 30 min, one oocyte was secured with a holding pipette (inner diameter=25-35 μm and outer diameter=80-100 μm). After being placed at the position of 5-6 o'clock, the first polar body and the adjacent cytoplasm (approx. 10% of the total volume of the oocyte) presumptively containing metaphase plate were aspirated and removed with a beveled injection pipette (inner diameter=20 μm). A fetal fibroblast cell was then injected into the space through the same slot. After nuclear transfer (NT), reconstructed couplets were transferred into drops of media covered with mineral oil for recovery for 1 to 1.5 hrs until fusion and activation was conducted. Reconstructed couplets were incubated in fusion medium for 4 min. Couplets were aligned manually using a finely pulled and polished glass capillary to make the contact plane parallel to electrodes. A single, 30 μsec, direct current pulse of 2.0 kV/cm was then applied. After culture in drops of PZM-3 medium supplemented with 7.5 μg/mL CB for 30-60 min, fusion results were examined under a stereomicroscope. Fused couplets were subjected to a second pulse in activation solution. After 30 min incubation in T10 they were transferred to PZM-3 medium to evaluate in vitro development.

Embryo Culture and Transfer

[0359] Reconstructed embryos were cultured in PZM-3 medium (Dobrinsky, J. T. et al. Biol Reprod 55, 1069-1074 (1996) supplemented with 4 mg/ml BSA. Zona-free embryos produced from HMC were cultured in the modified WOWs system (Feltrin, C. Et al. Reprod Fertil Dev 18, 126 (2006). Two different cell lines (LW1-2 for HMC, LW2 for TC) were used as nuclear donor cells for HMC and TC to allow the identification of the offspring from the two procedures. LW1-2 and LW2 originate from fetuses from a cross (with Duroc) and pure Danish landrace, respectively.

[0360] The average blastocyst per reconstructed embryo rate after in vitro culture for 7 days was 50.1±2.8% (mean±S.E.M), which is significantly higher (p<0.01) for HMC than that of TC performed in parallel in our laboratory (Table 9) and also the highest one that has ever been reported in pig cloning.

[0361]

TABLE 9
In vitro development of embryos produced from
handmade cloning and traditional cloning
		No. of
	Somatic cell	reconstructed	Cleavage	Blastocyst
Group	donor	embryos	rate (%)	rate (%)
HMC	LW1-2	643	83.7 ± 4.90a	50.06 ± 2.80a
TC	LW2	831	74.86 ± 13.16b	28.98 ± 2.84b
a,bValues of different superscripts within columns are significantly different (p < 0.05).
*mean ± S.E.M.

[0362] Mixed blastocysts produced from both HMC and TC were surgically transferred to 11 naturally synchronized sows on Day 4 or 5 of estrous cycle. Six (55%) recipients were diagnosed pregnant by ultrasonography, 2 aborted and by the time of writing 2 have delivered 3 and 10 piglets, respectively. A litter size of 10 cloned piglets is, according to our knowledge, the largest litter size so far achieved in pig cloning. All of them are healthy and behave normally except one showed rigid flexure of distal joint of one foreleg. %).

[0363] Preliminary results suggest that when embryos of similar stages were transferred, recipients on Day 4 of the estrous cycle supported pregnancy establishment better than those of Day 5 (Table 10).

[0364]

TABLE 10
In vivo development of cloned porcine embryos
	Embryos		No. of piglets born
	transferred	Embryo	Recipient		piglets	No.	Gestation
Recipient	HMC	TC	stage	cycle	Pregnancy	from	piglets	length
number	embryo	embryo	(Day)	(Day)	status	HMC	from TC	(Day)
1327	22	10	D 5, 6, 7	4	Y	2	1	116
1539	36	10	D 7	4	Y	8	2	115
1309	30	28	D 5, 6	4	Y
1553	45	44	D 5, 6	4	Y
1668	48	18	D 5, 6	5	Y, aborted
1428	78	22	D 5, 6	5	Y, aborted
1725	44	4	D 5, 6, 7	5	N	—	—	—
1643	22	11	D 5, 6, 7	4	N	—	—	—
1520	30	26	D 5, 6	4	N	—	—	—
1363	37	7	D 6, 7	5	N	—	—	—
1560	99	42	D 5, 6, 7	5	N	—	—	—

Microsatellite Analysis

[0365] Parental analysis using 10 different porcine microsatellite markers confirmed the identical genotype of cloned piglets and donor cells used for nuclear transfer. Identification was done by microsatellite analysis of genomic DNA from each of the newborn piglets, the surrogate sow, and the donor skin fibroblasts LW1-2 and LW2 originating from two fetuses that represent Danish landrace and Duroc, respectively. Ten polymorphic microsatellite loci (SW886, SW58, SW2116, SW1989, SW152, SW378, KS139, S0167, SW1987, SW957) located on different porcine chromosomes were amplified by 3-color multiplex PCR and the products analyzed on the Genetic Analyzer 3130 X1 (Applied Biosystems) using the program Gene Mapper 3.7.

[0366] For the second recipient, the offspring per embryo rate (22%) was the highest one ever reported so far in pig cloning (Walker, S. C. et al. Cloning Stem Cells 7, 105-112 (2005); Hoshino, Y. et al. Cloning Stem Cells 7, 17-26 (2005)). Comparable live birth/transferred embryo efficiencies were obtained in HMC (17%) and TC (15%).

Statistical Analysis

[0367] Differences between the experimental groups were evaluated using independent-samples t-test by SPSS 11.5. P<0.05 was considered significant.

Sequence Listing

[0368]

SEQ ID NO: 1: Sus scrofa ApoE gene sequence
1	ctcgagaggg agtgagggtt aaaactctgt ggtgcaacgg aaacgaatcc aactgggaaa
61	ccatgaggct gtgggttgga tccccggcct cgctcaatgg gttaaggatc cagcacggcg
121	ctgccgtgag ctgtggtgta ggtcgcagac gaagcttgga tcccacttgg ctgtggctgt
181	ggctgtggct gtggtgtagg cccgcagctg taactgtaat tcgaccccta gcctgggaac
241	ctccacaagc cacgggtgtg gccctaaaaa gcaaaaaaac gaaagcaaaa agaacactct
301	caaagcctaa actttgagca aaaagaacac tctcaaagcc taaactttga gcagatgcct
361	tacaccgccc ccacgcctct catccccttt ctgtctgggc ctccagctcc cttccccctt
421	aacccagaaa tcccagacct cagacccaag gatttcgaat ccccaggcct tggcccaatt
481	ctatcatccc agcacaggac aagaaaaaag cagggccggg ccttctggtc ctgctcctct
541	ccctgccagc ccaccccacc agtggcatgg aaaaagctcc ggaattactg ggtgaaaaaa
601	acctcttcca tgggggctgg aattaggggg ggggtgatgg ttgccaaccc cacccctccc
661	ctccctccct tcccccaccc tgctgtgtga aaggggaggc cagcccactt cgtgacccga
721	cgggggctgg cccagctggc cccagttctg gaggagtggg cggggcgggg ggagccctat
781	aattggccga atctgggctc cctgaatcat actcagcccc ggaggaggaa ggaggaagga
841	ggaggaggaa gcaaccggtg aggagcagac ctgggggcac agagatgggc tcggggcttc
901	ggtgtggggg ggtgggctgt cgggggagga ggaaatgacc tggccccccg gggccaccac
961	cgaggcagga gttggggatg aggctagagc ccagggactg gacctagaag gagggtgggc
1021	agcaggagga ggttatccgc cttggctgga aggggaggtc agggaagcag cgggacctgt
1081	aggaagaacc agacgagcca gagccgacga attgtactgg caggtatggc gcatctactc
1141	aagttttgag cacactaaga gctccatcga ggagacccag gggtggcggc gaccaggggt
1201	gacctcgacc gggctggcgg cagggtagct agagcgttgg tggaaggaca tgtaaatgag
1261	gattaaatta gggaatgagt ggaaaacagg gtttagatgt gaagttggag cttggaatgt
1321	gaaggtacca ggaagaacgt gagcttggag cccagaaagc aaggctgggg ctcacatggg
1381	actccagggt ggaaggggtg gggggcgacg tgggtggaat ttgaaccctg ggaaaaaagg
1441	aaggcttttg gccgcacccg acctggggat ggggagatag gagaagacaa tgagggaatt
1501	acacggacaa tggaaaggat ctgctcggga aatatctgct tggattaggc tgatgcagat
1561	aagggggtgc aaggcttgga aggctgtgac tggacagggc tgggctctgg gtgagaggag
1621	cgagccccgc cgctgttgag tgacaatttc tccctcctgc aggttggcca atcgcaagcc
1681	agaagatgag ggttctgtgg gttgctttgg tggtaaccct cctcgcaggt atgggggtgg
1741	ggcttgctca ggttccctgc ccctccccca tccccggtgc ccctccttca tccctgggtc
1801	tcttctgctg gtctctcttc cccttgagga gaggcctaga tgtgaggcct ctctggcact
1861	ccttgcttct gaacagctcg ttttactctc tgagcctcag tttccccatc tttaaaatgg
1921	gagttatgtt gagagattcc agctgtggct cagcaggtta agaacccgac tagtatccat
1981	gaggaagagg gttcaatccc ctggcttcgc tcagcgggtt aaggatccgg cgttgccatg
2041	agctgcggca taagtcgcag atgcagctcg aatcgggtgt tgctgtggct gtggtgcagg
2101	ctggcagcta tcgcttccat cggacccctc gcctgggaac ttccacgtat gccactggtg
2161	cagccctaaa agacaaacaa acaaaaacga aagaaagaga aaagaaagga aagggggctt
2221	ctgtttctaa tgcgttgttg cctggcaggg cgtgagcatt agatacgtgt cagctgtgac
2281	tagcgtgcac ggagcacaca atccatgctt gtccagtaat tagacaggct gggtgtcctt
2341	ccaccccctc cctgcccacc agtgctctag agaagcccac ccaccagggc tgggggagca
2401	cctgctctgt accaggtacc gtgtgctggg agggggcaga ggacctgatg gctgtgaact
2461	ggctcggtgc aggatgccgg acagaggacg agccggggcc gccgccggag gtgcacgtgt
2521	ggtgggagga gcccaagtgg cagggcagcc agccctggga gcaggccctg ggccgcttct
2581	gggattacct gcgctgggtg cagtccctgt ctgaccaagt gcaggaggag ctgctcagca
2641	ccaaggtcac ccaggaactg acgtaagtgc ccacccgact cccgccgcgc gcgcgcgcgc
2701	gcgcgcgcgc gcctgaccct cctggcgaac cgtgtgttct ggaccctcag gctccacccg
2761	tccgggtttc cttctgtcct tgtcgccaac tcttgggggt ctgggtctct gtttcttttt
2821	tttccttcct ccttttttgg ggggaaaaaa ctttttcttt tttctttcat ttgacttcat
2881	gtcttgcttt ctttccatct tgagctcctg ccttcgcctg tctctgggtc agtcttgccg
2941	tcccttgctg tctctgaatc tctggcacgt cctggccatc gccagctcag gagccctcct
3001	tctccccctc accgcccccg ccctctctgc gcccagggag ctgatagagg agagcatgaa
3061	ggaggtgaag gcctaccgcg aggagctgga ggcgcagctg ggccccgtga cccaggagac
3121	gcaggcgcgc ctgtccaagg agctgcaggc ggcgcaggcc cgcgtgggcg ccgacatgga
3181	ggacgtgcgc aaccgcttgg tgctctaccg cagcgaggtg cacaacatgt tgggccagac
3241	caccgaggag ctgcggagcc gcctggcttc ccacctgcgc aagctgcgca agcggctgct
3301	ccgcgacacc gaggacctgc agaagcgcct ggccgtgtac caggcggggc tgcgcgaggg
3361	cgccgagcgc agcgtgagcg ccctccgcga gcgcctcggg cccctggtgg agcagggccg
3421	attgcgcgcc gccaccctga gtaccagggc cggccagccg ctgcgcgagc gcgcggaagc
3481	ctggggccag aagctgcgcg gacggctgga ggagatgggc agccggaccc gcgaccgcct
3541	ggatgagatg cgtgagcagc tggaggaggt gcgcaccaaa gtggaggagc agggcagcca
3601	gttgcgcctg caggccgagg gattccacgc cctcctcaaa ggctggttcg agcctctggt
3661	ggaagacata cggcgccagt gggccgggct ggtggagagg atgcagtcgg gcgtgagcat
3721	aagctcctcc acctctgcgc ccagtgataa tcagtgagtg ccctctcatc cgggcacccc
3781	cttcggggcc ccgttcctgc ccaactcccc cgcctccccc agccttagat gccctcttgg
3841	tgggcccctg cttaataaag attcatcaag cttcacagca gcttctgggt gtccccggtg
3901	tgatttctca gctccagcct cagtttccct ttccttccct gcactgacca cccagttctc
3961	tgtcctgccc tctgcctgtg tgtgtctatt tgtctcttct cccccttttc tttttttttg
4021	gccgagccca tggcatgcgg aagttccccc ggccagggat tgaacccatg ccacagccgc
4081	cacaacgaag gatccttaac tactaggcca ccagggaact ccatcctttc taactctgtc
4141	tttgctttcc cttttttagc gttttagggc tgcaccctca gcatgtggaa gtccccaggc
4201	taggggtcaa attggcgcta cagctgccag cctacaccac agccccagca acgcaggatt
4261	cctcgag

ApoE Sus scrofa

[0369]

LOCUS	SSU70240 4267 bp DNA linear MAM 10-AUG-
	1998
DEFINITION	Sus scrofa apolipoprotein-E (Apo-E) gene, complete cds.
ACCESSION	U70240
VERSION	U70240.1 GI: 2388608
KEYWORDS	.
SOURCE	Sus scrofa (pig)
ORGANISM	Sus scrofa
	Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;
	Euteleostomi; Mammalia; Eutheria; Laurasiatheria;
	Cetartiodactyla; Suina; Suidae;
	Sus.
REFERENCE	1 (bases 1 to 4267)
AUTHORS	Ramsoondar, J. J., Rucker, E. B., Vasquez, J. C.,
	Gallagher, D. S., Grimm, D. R., Lunney, J. K., Schook,
	L. B. and Piedrahita, J. A.
TITLE	Isolation and genetic characterization of the porcine
	apolipoprotein E gene
JOURNAL	Anim. Genet. 29 (1), 43-47 (1998)
PUBMED	9682450
REFERENCE	2 (bases 1 to 4267)
AUTHORS	Ramsoondar, J. J. and Piedrahita, J. A.
TITLE	Direct Submission
JOURNAL	Submitted (10-SEP-1996) VAPH, Texas A&M University, College
	Station, TX 77843, USA
FEATURES	Location/Qualifiers
source	1 . . . 4267
	/organism=“Sus scrofa”
	/mol_type=“genomic DNA”
	/db_xref=“taxon:9823”
gene	832 . . . 3879
	/gene=“Apo-E”
mRNA	join(832 . . . 857, 1686 . . . 17282473 . . .
	2662, 3037 . . . 3879)
	/gene=“Apo-E”
exon	832 . . . 857
	/gene=“Apo-E”
	/number=1
intron	858 . . . 1662
	/gene=“Apo-E”
	/number=1
exon	1663 . . . 1728
	/gene=“Apo-E”
	/number=2
CDS	join(1686 . . . 1728, 2473 . . . 2662, 3037 . . . 3757)
	/gene=“Apo-E”
	/note=“plasma lipoprotein”
	/codon_start=1
	/product=“apolipoprotein-E”
	/protein_id=“AAC29512.1”
	/db_xref=“GI:2388609”
SEQ ID NO: 2: Sus scrofa ApoE protein
/translation=“MRVLWVALVVTLLAGCRTEDEPGPPPEVHVWWEEPKWQGSQPWE
QALGRFWDYLRWVQSLSDQVQEELLSTKVTQELTELIEESMKEVKAYREELEAQLGPV
TQETQARLSKELQAAQARVGADMEDVRNRLVLYRSEVHNMLGQTTEELRSRLASHLRK
LRKRLLRDTEDLQKRLAVYQAGLREGAERSVSALRERLGPLVEQGRLRAATLSTRAGQ
PLRERAEAWGQKLRGRLEEMGSRTRDRLDEMREQLEEVRTKVEEQGSQLRLQAEGFHA
LLKGWFEPLVEDIRRQWAGLVERMQSGVSISSSTSAPSDNQ”

[0370]

SEQ ID NO: 27: Homo sapiens ApoE protein sequence
LOCUS	AF261279 5491 bp DNA linear PRI
	27-OCT-2000
DEFINITION	Homo sapiens apolipoprotein-E gene, complete cds.
ACCESSION	AF261279
VERSION	AF261279.1 GI: 11034800
KEYWORDS	.
SOURCE	Homo sapiens (human)
ORGANISM	Homo sapiens
	Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;
	Euteleostomi;
	Mammalia; Eutheria; Euarchontoglires; Primates;
	Haplorrhini;
	Catarrhini; Hominidae; Homo.
REFERENCE	1 (bases 1 to 5491)
AUTHORS	Nickerson, D. A., Taylor, S. L., Fullerton, S. M., Weiss, K. M.,
	Clark, A. G., Stengard, J. H., Salomaa, V., Boerwinkle, E. and
	Sing, C. F.
TITLE	Sequence diversity and large-scale typing of SNPs in the
	human
	apolipoprotein E gene
JOURNAL	Genome Res. 10 (10), 1532-1545 (2000)
PUBMED	11042151
REFERENCE	2 (bases 1 to 5491)
AUTHORS	Nickerson, D. A.
TITLE	Direct Submission
JOURNAL	Submitted (27-APR-2000) Department of Molecular
	Biotechnology,
	University of Washington, Box 357730, Seattle, WA 98195,
	USA
FEATURES	Location/Qualifiers
source	1 . . . 5491
	/organism=“Homo sapiens”
	/mol_type=“genomic DNA”
	/db_xref=“taxon:9606”
	/chromosome=“19”
	/map=“19q13.2”
repeat_region	<3 . . . >108
	/note=“putative”
	/rpt_family=“MIR”
	/rpt_type=dispersed
variation	73
	/frequency=“0.01”
	/replace=“t”
repeat_region	<207 . . . >295
	/note=“putative”
	/rpt_family=“MIR”
	/rpt_type=dispersed
variation	308
	/frequency=“0.01”
	/replace=“t”
satellite	<320 . . . >339
	/note=“putative”
	/rpt_type=tandem
repeat_region	<340 . . . >637
	/note=“putative”
	/rpt_family=“Alu”
	/rpt_type=dispersed
variation	471
	/frequency=“0.01”
	/replace=“g”
variation	545
	/frequency=“0.01”
	/replace=“t”
variation	560
	/frequency=“0.22”
	/replace=“t”
variation	624
	/frequency=“0.07”
	/replace=“c”
satellite	<638 . . . >718
	/note=“putative”
	/rpt_type=tandem
variation	832
	/frequency=“0.45”
	/replace=“t”
mRNA	join(1060 . . . 1094, 1855 . . . 1920, 3013 . . . 3205,
	3786 . . . 4645)
	/product=“apolipoprotein-E”
variation	1163
	/frequency=“0.35”
	/replace=“c”
variation	1522
	/frequency=“0.01”
	/replace=“a”
variation	1575
	/frequency=“0.01”
	/replace=“t”
CDS	join(1878 . . . 1920, 3013 . . . 3205, 3786 . . . 4503)
	/note=“APOE”
	/codon_start=1
	/product=“apolipoprotein-E”
	/protein_id=“AAG27089.1”
	/db_xref=“GI:11034801”
/translation=“MKVLWAALLVTFLAGCQAKVEQAVETEPEPELRQQTEWQSGQRW
ELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTP
VAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLR
KLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAG
QPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQ
ARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH”
variation	1998
	/frequency=“0.10”
	/replace=“a”
repeat_region	<2124 . . . >2435
	/note=“putative”
	/rpt_family=“Alu”
	/rpt_type=dispersed
variation	2440
	/frequency=“0.21”
	/replace=“a”
repeat_region	<2569 . . . >2848
	/note=“putative”
	/rpt_family=“Alu”
	/rpt_type=dispersed
variation	2907
	/frequency=“0.01”
	/replace=“g”
variation	3106
	/frequency=“0.01”
	/replace=“c”
repeat_region	<3472 . . . >3588
	/note=“putative”
	/rpt_family=“Alu”
	/rpt_type=dispersed
variation	3673
	/frequency=“0.01”
	/replace=“g”
variation	3701{circumflex over ( )}3702
	/frequency=“0.01”
	/replace=“ct”
variation	3937
	/frequency=“0.14”
	/replace=“c”
variation	4036
	/frequency=“0.01”
	/replace=“t”
variation	4075
	/frequency=“0.07”
	/replace=“t”
repeat_region	<4755 . . . >5056
	/note=“putative”
	/rpt_family=“Alu”
	/rpt_type=dispersed
repeat_region	<5065 . . . >5476
	/note=“putative”
	/rpt_family=“Alu”
	/rpt_type=dispersed
variation	5229{circumflex over ( )}5230
	/frequency=“0.03”
	/replace=“gg”
variation	5229
	/frequency=“0.07”
	/replace=“t”
variation	5229{circumflex over ( )}5230
	/frequency=“0.40”
	/replace=“g”
variation	5230
	/frequency=“0.13”
	/replace=“”
variation	5361
	/frequency=“0.06”
	/replace=“c”
SEQ ID NO: 28: Homo sapiens ApoE gene sequence
1	cttgatgctc agagaggaca agtcatttgc ccaaggtcac acagctggca actggcagag
61	ccaggattca cgccctggca atttgactcc agaatcctaa ccttaaccca gaagcacggc
121	ttcaagcccc tggaaaccac aatacctgtg gcagccaggg ggaggtgctg gaatctcatt
181	tcacatgtgg ggagggggct cccctgtgct caaggtcaca accaaagagg aagctgtgat
241	taaaacccag gtcccatttg caaagcctcg acttttagca ggtgcatcat actgttccca
301	cccctcccat cccacttctg tccagccgcc tagccccact ttcttttttt tctttttttg
361	agacagtctc cctcttgctg aggctggagt gcagtggcga gatctcggct cactgtaacc
421	tccgcctccc gggttcaagc gattctcctg cctcagcctc ccaagtagct aggattacag
481	gcgcccgcca ccacgcctgg ctaacttttg tatttttagt agagatgggg tttcaccatg
541	ttggccaggc tggtctcaaa ctcctgacct taagtgattc gcccactgtg gcctcccaaa
601	gtgctgggat tacaggcgtg agctaccgcc cccagcccct cccatcccac ttctgtccag
661	ccccctagcc ctactttctt tctgggatcc aggagtccag atccccagcc ccctctccag
721	attacattca tccaggcaca ggaaaggaca gggtcaggaa aggaggactc tgggcggcag
781	cctccacatt ccccttccac gcttggcccc cagaatggag gagggtgtct ggattactgg
841	gcgaggtgtc ctcccttcct ggggactgtg gggggtggtc aaaagacctc tatgccccac
901	ctccttcctc cctctgccct gctgtgcctg gggcaggggg agaacagccc acctcgtgac
961	tgggggctgg cccagcccgc cctatccctg ggggaggggg cgggacaggg ggagccctat
1021	aattggacaa gtctgggatc cttgagtcct actcagcccc agcggaggtg aaggacgtcc
1081	ttccccagga gccggtgaga agcgcagtcg ggggcacggg gatgagctca ggggcctcta
1141	gaaagagctg ggaccctggg aagccctggc ctccaggtag tctcaggaga gctactcggg
1201	gtcgggcttg gggagaggag gagcgggggt gaggcaagca gcaggggact ggacctggga
1261	agggctgggc agcagagacg acccgacccg ctagaaggtg gggtggggag agcagctgga
1321	ctgggatgta agccatagca ggactccacg agttgtcact atcatttatc gagcacctac
1381	tgggtgtccc cagtgtcctc agatctccat aactggggag ccaggggcag cgacacggta
1441	gctagccgtc gattggagaa ctttaaaatg aggactgaat tagctcataa atggaacacg
1501	gcgcttaact gtgaggttgg agcttagaat gtgaagggag aatgaggaat gcgagactgg
1561	gactgagatg gaaccggcgg tggggagggg gtggggggat ggaatttgaa ccccgggaga
1621	ggaagatgga attttctatg gaggccgacc tggggatggg gagataagag aagaccagga
1681	gggagttaaa tagggaatgg gttgggggcg gcttggtaaa tgtgctggga ttaggctgtt
1741	gcagataatg caacaaggct tggaaggcta acctggggtg aggccgggtt ggggccgggc
1801	tgggggtggg aggagtcctc actggcggtt gattgacagt ttctccttcc ccagactggc
1861	caatcacagg caggaagatg aaggttctgt gggctgcgtt gctggtcaca ttcctggcag
1921	gtatgggggc ggggcttgct cggttccccc cgctcctccc cctctcatcc tcacctcaac
1981	ctcctggccc cattcaggca gaccctgggc cccctcttct gaggcttctg tgctgcttcc
2041	tggctctgaa cagcgatttg acgctctctg ggcctcggtt tcccccatcc ttgagatagg
2101	agttagaagt tgttttgttg ttgttgtttg ttgttgttgt tttgtttttt tgagatgaag
2161	tctcgctctg tcgcccaggc tggagtgcag tggcgggatc tcggctcact gcaagctccg
2221	cctcccaggt ccacgccatt ctcctgcctc agcctcccaa gtagctggga ctacaggcac
2281	atgccaccac acccgactaa cttttttgta ttttcagtag agacggggtt tcaccatgtt
2341	ggccaggctg gtctggaact cctgacctca ggtgatctgc ccgtttcgat ctcccaaagt
2401	gctgggatta caggcgtgag ccaccgcacc tggctgggag ttagaggttt ctaatgcatt
2461	gcaggcagat agtgaatacc agacacgggg cagctgtgat ctttattctc catcaccccc
2521	acacagccct gcctggggca cacaaggaca ctcaatacat gcttttccgc tgggcgcggt
2581	ggctcacccc tgtaatccca gcactttggg aggccaaggt gggaggatca cttgagccca
2641	ggagttcaac accagcctgg gcaacatagt gagaccctgt ctctactaaa aatacaaaaa
2701	ttagccaggc atggtgccac acacctgtgc tctcagctac tcaggaggct gaggcaggag
2761	gatcgcttga gcccagaagg tcaaggttgc agtgaaccat gttcaggccg ctgcactcca
2821	gcctgggtga cagagcaaga ccctgtttat aaatacataa tgctttccaa gtgattaaac
2881	cgactccccc ctcaccctgc ccaccatggc tccaaagaag catttgtgga gcaccttctg
2941	tgtgccccta ggtactagat gcctggacgg ggtcagaagg accctgaccc accttgaact
3001	tgttccacac aggatgccag gccaaggtgg agcaagcggt ggagacagag ccggagcccg
3061	agctgcgcca gcagaccgag tggcagagcg gccagcgctg ggaactggca ctgggtcgct
3121	tttgggatta cctgcgctgg gtgcagacac tgtctgagca ggtgcaggag gagctgctca
3181	gctcccaggt cacccaggaa ctgaggtgag tgtccccatc ctggcccttg accctcctgg
3241	tgggcggcta tacctcccca ggtccaggtt tcattctgcc cctgtcgcta agtcttgggg
3301	ggcctgggtc tctgctggtt ctagcttcct cttcccattt ctgactcctg gctttagctc
3361	tctggaattc tctctctcag ctttgtctct ctctcttccc ttctgactca gtctctcaca
3421	ctcgtcctgg ctctgtctct gtccttccct agctctttta tatagagaca gagagatggg
3481	gtctcactgt gttgcccagg ctggtcttga acttctgggc tcaagcgatc ctcccgcctc
3541	ggcctcccaa agtgctggga ttagaggcat gagccacctt gcccggcctc ctagctcctt
3601	cttcgtctct gcctctgccc tctgcatctg ctctctgcat ctgtctctgt ctccttctct
3661	cggcctctgc cccgttcctt ctctccctct tgggtctctc tggctcatcc ccatctcgcc
3721	cgccccatcc cagcccttct ccccgcctcc cactgtgcga caccctcccg ccctctcggc
3781	cgcagggcgc tgatggacga gaccatgaag gagttgaagg cctacaaatc ggaactggag
3841	gaacaactga ccccggtggc ggaggagacg cgggcacggc tgtccaagga gctgcaggcg
3901	gcgcaggccc ggctgggcgc ggacatggag gacgtgtgcg gccgcctggt gcagtaccgc
3961	ggcgaggtgc aggccatgct cggccagagc accgaggagc tgcgggtgcg cctcgcctcc
4021	cacctgcgca agctgcgtaa gcggctcctc cgcgatgccg atgacctgca gaagcgcctg
4081	gcagtgtacc aggccggggc ccgcgagggc gccgagcgcg gcctcagcgc catccgcgag
4141	cgcctggggc ccctggtgga acagggccgc gtgcgggccg ccactgtggg ctccctggcc
4201	ggccagccgc tacaggagcg ggcccaggcc tggggcgagc ggctgcgcgc gcggatggag
4261	gagatgggca gccggacccg cgaccgcctg gacgaggtga aggagcaggt ggcggaggtg
4321	cgcgccaagc tggaggagca ggcccagcag atacgcctgc aggccgaggc cttccaggcc
4381	cgcctcaaga gctggttcga gcccctggtg gaagacatgc agcgccagtg ggccgggctg
4441	gtggagaagg tgcaggctgc cgtgggcacc agcgccgccc ctgtgcccag cgacaatcac
4501	tgaacgccga agcctgcagc catgcgaccc cacgccaccc cgtgcctcct gcctccgcgc
4561	agcctgcagc gggagaccct gtccccgccc cagccgtcct cctggggtgg accctagttt
4621	aataaagatt caccaagttt cacgcatctg ctggcctccc cctgtgattt cctctaagcc
4681	ccagcctcag tttctctttc tgcccacata ctggccacac aattctcagc cccctcctct
4741	ccatctgtgt ctgtgtgtat ctttctctct gccctttttt ttttttttag acggagtctg
4801	gctctgtcac ccaggctaga gtgcagtggc acgatcttgg ctcactgcaa cctctgcctc
4861	ttgggttcaa gcgattctgc tgcctcagta gctgggatta caggctcaca ccaccacacc
4921	cggctaattt ttgtattttt agtagagacg agctttcacc atgttggcca ggcaggtctc
4981	aaactcctga ccaagtgatc cacccgccgg cctcccaaag tgctgagatt acaggcctga
5041	gccaccatgc ccggcctctg cccctctttc ttttttaggg ggcagggaaa ggtctcaccc
5101	tgtcacccgc catcacagct cactgcagcc tccacctcct ggactcaagt gataagtgat
5161	cctcccgcct cagcctttcc agtagctgag actacaggcg cataccacta ggattaattt
5221	gggggggggg tggtgtgtgt ggagatgggg tctggctttg ttggccaggc tgatgtggaa
5281	ttcctgggct caagcgatac tcccaccttg gcctcctgag tagctgagac tactggctag
5341	caccaccaca cccagctttt tattattatt tgtagagaca aggtctcaat atgttgccca
5401	ggctagtctc aaacccctgg gctcaagaga tcctccgcca tcggcctccc aaagtgctgg
5461	gattccaggc atgggctccg agcggcctgc c
SEQ ID NO: 29: Homo sapiens ApoE gene
1	gggatccttg agtcctactc agccccagcg gaggtgaagg acgtccttcc ccaggagccg
61	actggccaat cacaggcagg aagatgaagg ttctgtgggc tgcgttgctg gtcacattcc
121	tggcaggatg ccaggccaag gtggagcaag cggtggagac agagccggag cccgagctgc
181	gccagcagac cgagtggcag agcggccagc gctgggaact ggcactgggt cgcttttggg
241	attacctgcg ctgggtgcag acactgtctg agcaggtgca ggaggagctg ctcagctccc
301	aggtcaccca ggaactgagg gcgctgatgg acgagaccat gaaggagttg aaggcctaca
361	aatcggaact ggaggaacaa ctgaccccgg tggcggagga gacgcgggca cggctgtcca
421	aggagctgca ggcggcgcag gcccggctgg gcgcggacat ggaggacgtg tgcggccgcc
481	tggtgcagta ccgcggcgag gtgcaggcca tgctcggcca gagcaccgag gagctgcggg
541	tgcgcctcgc ctcccacctg cgcaagctgc gtaagcggct cctccgcgat gccgatgacc
601	tgcagaagcg cctggcagtg taccaggccg gggcccgcga gggcgccgag cgcggcctca
661	gcgccatccg cgagcgcctg gggcccctgg tggaacaggg ccgcgtgcgg gccgccactg
721	tgggctccct ggccggccag ccgctacagg agcgggccca ggcctggggc gagcggctgc
781	gcgcgcggat ggaggagatg ggcagccgga cccgcgaccg cctggacgag gtgaaggagc
841	aggtggcgga ggtgcgcgcc aagctggagg agcaggccca gcagatacgc ctgcaggccg
901	aggccttcca ggcccgcctc aagagctggt tcgagcccct ggtggaagac atgcagcgcc
961	agtgggccgg gctggtggag aaggtgcagg ctgccgtggg caccagcgcc gcccctgtgc
1021	ccagcgacaa tcactgaacg ccgaagcctg cagccatgcg accccacgcc accccgtgcc
1081	tcctgcctcc gcgcagcctg cagcgggaga ccctgtcccc gccccagccg tcctcctggg
1141	gtggacccta gtttaataaa gattcaccaa gtttcacgca aaaaaaaaaa aaaaaaaaaa
1201	aaaaaaaaaa aaaaaaaaaa aaa

SEQ ID NO: 3
LDL Receptor cDNA Sequence

[0371]

Sus scrofa low density lipoprotein receptor (LDLR) mRNA, LDLR-N
allele, partial cds.
ACCESSION	AF065990
VERSION	AF065990.1 GI: 3153894
KEYWORDS	.
SOURCE	Sus scrofa (pig)
ORGANISM	Sus scrofa
	Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;
	Euteleostomi;
	Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla;
	Suina; Suidae;
	Sus.
REFERENCE	1 (bases 1 to 2403)
AUTHORS	Hasler-Rapacz, J., Ellegren, H., Fridolfsson, A. K.,
	Kirkpatrick, B.,
	Kirk, S., Andersson, L. and Rapacz, J.
TITLE	Identification of a mutation in the low density
	lipoprotein
	receptor gene associated with recessive familial
	hypercholesterolemia in swine
JOURNAL	Am. J. Med. Genet. 76 (5), 379-386 (1998)
PUBMED	9556295
REFERENCE	2 (bases 1 to 2403)
AUTHORS	Hasler-Rapacz, J., Ellegren, H., Fridolfsson, A. K.,
	Kirkpatrick, B.,
	Kirk, S., Andersson, L. and Rapacz, J.
TITLE	Direct Submission
JOURNAL	Submitted (16-MAY-1998) Genetics & Animal Science,
	University of
	Wisconsin, 1675 Observatory Drive, Madison, WI 53706, USA
FEATURES	Location/Qualifiers
source	1 . . . 2403
	/organism=“Sus scrofa”
	/mol_type=“mRNA”
	/db_xref=“taxon:9823”
	/chromosome=“2”
	/map=“2q; near the centromere”
	/tissue_type=“liver”
gene	<1 . . . >2403
	/gene=“LDLR”
	/allele=“N (normal)”
CDS	<1 . . . >2403
	/gene=“LDLR”
	/allele=“N (normal)”
	/note=“LDL-receptor; normolipidemic”
	/codon_start=1
	/product=“low density lipoprotein receptor”
	/protein_id=“AAC17444.1”
	/db_xref=“GI:3153895”
1	ttccagtgcc aagacgggaa atgcatctcc tacaagtgga tttgtgatgg gaacaccgag
61	tgcaaggacg ggtccgatga gtccctggag acgtgcatgt ctgtcacctg caagataggg
121	gactttagct gtgggggccg tgtcaaccgc tgcattcctg agtcttggag gtgtgacggt
181	cagcaggact gcgagaatgg ctcagatgag gaaggctgtt cccccaagac gtgctcccaa
241	gatgagttcc gctgccagga cggcaagtgc atcgccccaa agtttgtctg tgactcggac
301	cgggactgcc tggacggctc ggatgaagca tcctgcccca cacccacctg tggccccgcc
361	agcttccagt gcaacagctc cacctgcatc cctgagctgt gggcctgtga tggtgatcct
421	gactgcgagg acggctcaga cgagtggcca cagcactgca ggagccacag ctcatcactc
481	cccgagagga gcaacaaccc ctgctcagcc ctcgagttcc actgccacag tggcgagtgc
541	atccactcca gctggcgctg cgacggagac actgactgca aggacaagtc tgacgaggag
601	aactgcgatg tggccacgtg ccggcctgac gagttccagt gctcagacgg gacctgcatc
661	catggtagcc ggcagtgcga cagggaatat gactgcaagg acatgagcga cgagcagggc
721	tgtgtcaatg cgactctgtg cgaggggccc aacaagttca agtgtcaaag cggcgagtgc
781	atctccttgg acaaagtgtg caactcagtc agggactgcc gggactggtc agacgagccc
841	ctcaaggagt gtgggaccaa cgagtgtctg gacaacaagg gtggctgctc ccatatctgc
901	aatgacctca agatcggcta tgagtgcctc tgtcccgagg gcttccagct ggtggataag
961	cacagatgcg aagatatcga cgagtgtcag gacccagacg cctgcagcca gatctgcgtg
1021	aacctcgagg gcagctacaa gtgccagtgt gaggagggct tccagctgga gcctctcacc
1081	aaggcctgca aggccatagg caccatcgcc tacctcttct tcaccaaccg ccacgaggtg
1141	aggaagatga ccctggaccg tagtgagtac accagcctca tccccaacct gaagaacgtg
1201	gtcgctctgg acactgaggt ggccagcaat agaatctact ggtctgacct gtctcagagg
1261	aagatctaca gtacccagat cgacagggcc cccagctttt cctcctatga caccattatt
1321	ggcgaagatc tccaggcccc cgatgggctg gcggtggact ggatccacag caacatatac
1381	tggactgact ccatcctggg cactgtctcc gtggctgaca ccaagggcgt gaagaggaag
1441	actctcttcc aagagaaagg ctccaagcca cgggccattg tggtggaccc tgtccatggc
1501	ttcatgtact ggactgattg gggaaccccc gccaagatca agaagggcgg cctgaacgga
1561	gtggacgtct actcgctggt gacggaggac atccagtggc ccaatggcat caccctggat
1621	ctttctggcg gccgccttta ctgggtcgac tccaagctcc actccatctc cagcatcgat
1681	gtcaacgggg ggaaccggaa gaccgtcctg gaggacaaga cgaagctggc gcaccccttc
1741	tccttggcca tttttgagga taaagtattt tggacagata taatcaacga agccattttc
1801	agtgccaacc gcctcacagg ctcggacata catttgatgg cagaaaacct gttgtctcca
1861	gaggacattg tccttttcca caacctcaca cagccgagag gggtgaactg gtgtgaaagg
1921	accgccctcc aaaacggtgg ctgccagtac ctgtgtctgc cagctccaca gatcaaccca
1981	cgctcgccga agttcacctg tgcctgcccg gatggcatgc tgttggccaa ggacatgagg
2041	agctgtctca cagagactga acctgcagga accacccagg gaccttccat ggtcaactcg
2101	acagctgtgg ggccaaagca caccgccagc tctgagctca ccacagccga gtcagtgacg
2161	atgtcccaac atgccctggg cgacgttgct ggccgaggag tcactgagaa gccccagagc
2221	gtgggtgctc tgtacattgt cctccccatt gcactgctca tcctcctctt cttcggaacc
2281	ttcctcctct ggaagaactg gaggcttaag agcatcaaca gcattaactt cgacaaccct
2341	gtgtaccaga agaccacgga agacgaggtc cacatctgcc gcagccagga cggctacacc
2401	tac

SEQ ID NO: 4
Porcine LDL Amino Acid Sequence

[0372]

FQCQDGKCISYKWICDGNTECKDGSDESLETCMSVTCKIGDFSC

GGRVNRCIPESWRCDGQQDCENGSDEEGCSPKTCSQDEFRCQDGKCIAPK

FVCDSDRDCLDGSDEASCPTPTCGPASFQCNSSTCIPELWACDGDPDCED

GSDEWPQHCRSHSSSLPERSNNPCSALEFHCHSGECIHSSWRCDGDTDCK

DKSDEENCDVATCRPDEFQCSDGTCIHGSRQCDREYDCKDMSDEQGCVNA

TLCEGPNKFKCQSGECISLDKVCNSVRDCRDWSDEPLKECGTNECLDNKG

GCSHICNDLKIGYECLCPEGFQLVDKHRCEDIDECQDPDACSQICVNLEG

SYKCQCEEGFQLEPLTKACKAIGTIAYLFFTNRHEVRKMTLDRSEYTSLI

PNLKNVVALDTEVASNRIYWSDLSQRKIYSTQIDRAPSFSSYDTIIGEDL

QAPDGLAVDWIHSNIYWTDSILGTVSVADTKGVKRKTLFQEKGSKPRAIV

VDPVHGFMYWTDWGTPAKIKKGGLNGVDVYSLVTEDIQWPNGITLDLSGG

RLYWVDSKLHSISSIDVNGGNRKTVLEDKTKLAHPFSLAIFEDKVFWTDI

INEAIFSANRLTGSDIHLMAENLLSPEDIVLFHNLTQPRGVNWCERTALQ

NGGCQYLCLPAPQINPRSPKFTCACPDGMLLAKDMRSCLTETEPAGTTQG

PSMVNSTAVGPKHTASSELTTAESVTMSQHALGDVAGRGVTEKPQSVGAL

YIVLPIALLILLFFGTFLLWKNWRLKSINSINFDNPVYQKTTEDEVHICR

SQDGYTY

SEQ ID NO: 30: Human LDL receptor genomic sequence Chromosome 19. Coding sequence underlined

[0373]

1	gccccgagtg caatcgcggg aagccagggt ttccagctag gacacagcag gtcgtgatcc
61	gggtcgggac actgcctggc agaggctgcg agcatggggc cctggggctg gaaattgcgc
121	tggaccgtcg ccttgctcct cgccgcggcg gggactgcag gtaaggcttg ctccaggcgc
181	cagaataggt tgagagggag cccccggggg gcccttggga atttattttt ttgggtacaa
241	ataatcactc catccctggg agacttgtgg ggtaatggca cggggtcctt cccaaacggc
301	tggagggggc gctggagggg ggcgctgagg ggagcgcgag ggtcgggagg agtctgaggg
361	atttaaggga aacggggcac cgctgtcccc caagtctcca cagggtgagg gaccgcatct
421	tctttgagac ggagtctagc tctgtcgccc aggatggagt gcagtggcac gatctcagct
481	cactgcaacc tccgcctccc gggtttaagc gagtctcctc tctcagcctc ccgaatagct
541	gggattacag gcgcccaacc accacgcccg cctaattttt gtatttttag tagagacggg
601	ttttcaccat tttggccagg ctggtctcga accccgacct caggtgatct gcccaaaagt
661	gctgggatta caggcgtcag ccaccgcgcc cggccgggac cctctcttct aactcggagc
721	tgggtgtggg gacctccagt cctaaaacaa gggatcactc ccacccccgc cttaagtcct
781	tctgggggcg agggcgactg gagacccgga tgtccagcct ggaggtcacc gcgggctcag
841	gggtcccgat ccgctttgcg cgaccccagg gcgccactgc catcctgagt tgggtgcagt
901	cccgggattc cgccgcgtgc tccgggacgg gggccacccc ctcccgcccc tgcccccgcc
961	cctttggccc gccccccgaa ttccattggg tgtagtccaa caggccaccc tcgagccact
1021	ccccttgtcc aatgtgaggc ggtggaggcg gaggcgggcg tcgggaggac ggggcttgtg
1081	tacgagcggg gcggggctgg cgcggaagtc tgagcctcac cttgtccggg gcgaggcgga
1141	tgcaggggag gcctggcgtt cctccgcggt tcctgtcaca aaggcgacga caagtcccgg
1201	gtccccggag ccgcctccgc gacatacacg agtcgccctc cgttatcctg ggccctcctg
1261	gcgaagtccc cggtttccgc tgtgctctgt ggcgacacct ccgtccccac cttgtcctgg
1321	ggggcgccct cgccccacca gccccgatca agttcacaga ggggcccccg gccaccctca
1381	aggcctcggt tccttacgag gttgaaacgt tgcctcagaa tctccccgcc cctccttggt
1441	ctgcagccga gatcttcagc cacggtgggg cagctatccc ccgggaccga ccccctgggg
1501	tggcctcgct tcttcagagg ctgtgaatgg cttcggttca gctgtccaag cggcgatttt
1561	tcctctgggt gaaatggatt agattttaga tttccacaag aggctggtta gtgcatgatc
1621	ctgagttaga gctttttagg tggctttaaa ttagttgcag agagacagcc tcgccctaga
1681	caacagctac atggcccttt ccctcctgag aaccagccta gcctagaaaa ggattgggat
1741	tgcctgatga acacaaggat tgcaggaaac ttttttttta attggcaagg gggttggctt
1801	tgactggatg gagagctttg aactgccttg aaattcacgc tgtaactaac acaccagttt
1861	cctctgggag gccagagagg gagggagggt gtaatgaaat acggatgatt gttcttttat
1921	ttttatttac ttatttattt tttaactttt tgtagagatg aggtctcgct tggttgctca
1981	ggctggtctt gaactcctgg cctcaagcga tcctcctacc tcagcctccc aaagtgttgg
2041	gattacagga gtgagccacc gcgccccacc ggggatgatg atgattgcaa acattctgcc
2101	actcagtttt acaaaagaaa gagaggcact ggattaatgt gtatctcact caccaatcaa
2161	cctcttcctt aagagaaaat gttaaggaag tcttaggcaa ggccttgttt gttcatcact
2221	ttagtttctc tctcccggga tggctgagaa tgtgatgttt cctctgttgt caaggagact
2281	acacccctga tgttttcctc cagacttctg agagctggtg tgtgtttcta gcactttcta
2341	gctgcaccac ctcacgctgt agctggcttc aaggcatatc caggggggag tttcttgtcc
2401	atttccttta caaagggaag ttgttggaat ctgaaccgca agccttcact tagaccaaaa
2461	tcaggcaaca gcggtgagcg cagctccaaa cgtgtcaatg actcacccaa atttgagtaa
2521	gggagttggc tgctttaacg agccgcaggg tgattccctt gtcatttccg gaaataccta
2581	tcttccaggg aacactggga aaaaacaggg agacctttgt tgagacagaa aacctgtagg
2641	ggaattctgt tcctcattcc tgctcttatc tgtagacttc ctccctgata agatccaatt
2701	ctagatgggt cggttgctcc ttgctttgat gggtgctttg atgggcttta ttattattat
2761	tattattatt attattattt tgatgggctt tttgatgtcc cttttccttc cacactctgt
2821	cccaactgtc aagcaaatag ccttttgttg ctaagagact gcagatgtaa ccgaccagca
2881	gcaaacagtg agtcaggctc tctcttccgg aagcaaaatc aattgctgag atcactctgg
2941	ggaaaatacc caccttattt ggaaagaagc actgatcaat tgatgtctat tttttttttt
3001	tttgagttgg agtctcgccc tgtcacccag gctggagtgc aatggcataa tctcgcctca
3061	ctgcaatccc cgcctcccgg gttccagcaa ttctcctgcc tcagcctcct gagtagctgg
3121	aattataggc gcctgccaca acacccggct aatttttgta tttgtagtag agatggggtt
3181	tcaccacgtt ggccaggctg gtctcgaact cctgacctcg tgatccaccc gcctcagcct
3241	cccaaagtcc aaggattgca ggcgtgaccc actgtgccag ccaatcaatt gatttctcat
3301	tcattttcag ctggctctgt tcccttaagc caggggattt tcgtttgttt gtttcccctt
3361	caaggaaatg attctagcta cagttttgat ttccttgtac aactgttttc agtagcacag
3421	ggaaagaaaa catcgaaagc attcaccacc tcatttgtgt gctgggggaa aaagcagaaa
3481	tgtgtattct ctttttttgt ttcgatgacc ttgttcctga cttgttactc gtgacttgag
3541	agatcagagg gctagaggac tagaatttat agaggtgttt tttttgtttg tttatttttg
3601	ttcgagttgc ccaggctgga gtgcagtggc gcaatctcgg ctcactgcaa cctctgcctc
3661	ccaggttcaa gcgattcttc ggcctcagcc tcctgagtag ctggaactac aggcgcccgc
3721	caccacaccc agctaatttt tgtatttttc agtagagatg ggatttcacc atattggtca
3781	agctggcctc gaactcctga cctcgtgatc cacccgcctc agtttcccaa agtgctggga
3841	gtacaggcgt gagccgccgt gcccggcctt tttgtgtttt tgtgtttttg agaggagctc
3901	attgcttttt aggcttccct agcgtgagaa aatctgggga tccatgctct agtttacttc
3961	cttttttttt ttttttttga gatggagtct cgcttagatt gcctaatctc agctcattgc
4021	aacttctgcc tccggggttc aagggattct cgtgtctcag cctcctgggt agctaggata
4081	cgggcacccg ctaccatgcc tggctaattt tgtactttta gtagagacag ggtttcgcca
4141	cgttggccag gctggtctcg aactcctgac ctcaggtgag ccgcctgcct tggcctccca
4201	aagtgctgag attacaggcg tgagccaccg cgcttggcct aatttgcttt tcctgaaatt
4261	caaatggtct aatatgaaaa acgccaacct tgcttgaaag aataagaaag aggtgcggtt
4321	tcgttgggcc gttgatgttt ggaacaggac tggttttgtc cccttgctcg gaaagggcag
4381	caactgtgag gacagctccc tgacgtgctc tcactcagca ctgttccgtt cctgagcact
4441	gtccccacta gctaggccaa gggagctcat ttggcaggca actgctgtct ggctgcgcct
4501	gtggcagtaa aatctgcctt tattttttgg aggcagggtc ttgccctgtc gctcaggctg
4561	aagtgtgcag ttatagctca ctgcagcctc cagcttctgt actcaactga tcctcctctc
4621	tcagcctcct gagtagctgg gactatacgc acgtgttacc actcccacct cagtttgttt
4681	gtttatttat ttatttattt atttattgag atggagtttt gctcttgctg cccaggctgg
4741	agtgcaatgg cgcgatctcg gctcaccgca acctccacct cctggttcaa gcgattctcc
4801	tgcctcagcc tcctgagtag ctgggattac aggcatgcac caccacgccc ggctaatttt
4861	gtatttttcg tagagatggg gtttctccac attggttcag gctgttctcg aactcccaac
4921	ctcaggtgat ccacccgcct cagcctccca aagtgctggg attataggcg tgagcccccg
4981	aacccggcca ctcccagcta agtttaaatt ttttgtttgt ttgttcgttt gtttttattt
5041	tttgagacag agtctcccgc ccaggctgga gcgcagatca ctgcatcctt gacctcccag
5101	gcttaagcca tcctccccac tcagcctccc aagtagctgg gattacaggt gtgtgccact
5161	atgcttggct aagttgtgta ttttttgtag agatggggtt caagggattc tcgctttgtt
5221	gcctcggttg gtctcaaact cctgggctca agcagtcctc cctcctcagc ctcccaaggt
5281	gctggggaaa tccacttttg aaacattgtc tggagagttg cccaggtggt agatcacaga
5341	aataggtcat cgtggggtcc ttcccatggg tgcagtcttg agccacctgt ggccagcaaa
5401	tatttggaga ataatagtca ggggagagct tgaggtccag ggaaaggttt tgtttttctt
5461	cagggaaagg tttttattgt tctttatccc tccttaaagg accttcaggt gttactgaca
5521	ttcccggtct acccagtggc acatttagtt tgtaagctgg gccctcgtac agaggtaggg
5581	aggtgagagc attggattag tggtcaccaa agctgcggtc acctagtggg gtgatcagag
5641	gctcctccct taagatcttg attgccaacg cctctggccc aactttcctt tttatttatc
5701	gcaagcctcc tggaatctca attgcttttt gcccacccgg tgtgtcagca caagaaatga
5761	gtcatttcct cctttaagca cagttgaaat tgagctgtga gtcagtgagg tgtgtacgat
5821	attgtcaaag cggggtgtgt acagtattga cagatctgta gttgggcaag agaattatca
5881	gagtttgtga ccacagcaga ttccaaagct cgactcattt tcttctctct tccttccctt
5941	ttttcttttc tttttttttt tttttttgac agagtctcgc tctgttgccc aggctggagt
6001	gcagtggcac aatctgggct cactgcagcc cctgcctcct gggttcaaat gattctcatg
6061	tttcagcctc ccgagtagct gcaattacag gcattcgggt tcaagtgatt ctcctgcctc
6121	agccacctga gcagctggga ttacaggcgc ccgccaccac gcccggctaa tttttgtatt
6181	tttagtagag acggggtttc accatgttgg ccaggctggt ctcgaactcc tgaactcagg
6241	tgatccgccc acttcggcct cccaaagtgc tgagattaca gacgtgagtc accgcgccca
6301	gcctgttctg ttctttaatt ctcaaaacac cctctaggaa gtagagactg ccattctccc
6361	ccattttaca gatcaggaaa ctgagtccca gaaggattta gtcagttacc caagttgttc
6421	tagttaaatg gcctggaaag ccagtgaagc ccaggattgt ctatctaacc cccttactac
6481	tctaactttc agggaatcca catgaatgtg ctgggtcaac catcaaagtt gaaatggata
6541	aagggggctg gatgcggtgg ctgatgcctg taatcctagc actttgggag gccgagatgg
6601	gtgggtggat tgcttgagcc caagagtttg agaccagcct gggcaacata gtgagacacc
6661	tgtctctgca aaaaataaat aaaaagttag ctgagtgtga tggtgcaccc ctctagtcac
6721	agctgttgag ttaggcttag gcaggaggat cgcatgaacc tgggaggtgg aggcggccgt
6781	gagcctcagt catgccactg cactccaacc tgggcaacag agtgaaagcc ggtgtccgaa
6841	agagaaagaa aaaaagacat agatacatct tttaaagtta ggttgtatgt taattaccta
6901	caactcagtt tcaactgtgc ttaaaggagg aaatgactca tttcttgcta catatcaaat
6961	tagcccaaaa tgtagtggct taaaacaaca catttatgat ttctcagttt ttgcgtgtca
7021	ggaatttgga agcagcacag ctagacggtt ccagctcagg gtctctcatg aagttgcaat
7081	caaaatattg gcaggagaga aaaacatatt ttcagaagct gcaggcatag gaagacttgg
7141	ctggggttga aggatccact tccaagatgg cgcactcagt ggctcttggc tggaggcctc
7201	agttccctgc tgcgtggagc tctccctcca gctgcttgag tggactcatg acatgcagct
7261	ggcctcccct ggagcagtcg atccaacaat gagcatggcc atgaactagg ctcagaagcc
7321	actccctgtc gtctctacat tttcctatca gaagcaagtc attaaaagtc cagtgccact
7381	ccaggggaga cgaattaggc tctgccttct gaaaggatta tcacagaaga tgcggtccta
7441	tattcttttt ttaaaattat tctttttttt attttgtaga gatggggtct tggtatgttg
7501	cctaggccag tctggaattc ctgggctcaa acaatcctgt ctctgcctcc caaagtgttg
7561	ggattacagg catgagccac tgcacctggt catgtggtca tattttcttt ttcttttttt
7621	tttttttttg agacagagtc tctgtcgccc aggctggagt atggtggcgt gatctcagtt
7681	cactgcagcc tccgcctccc gggttcaagc gattctcctg cctcagcctc ctgagtagct
7741	gggattacag gcgcccgcca acatgcccag ctaatttttt tagtagagat ggggtttcac
7801	catgttagcc aggatggtct cgatctcctg atttggtgat ccgcccacct tggcctccca
7861	aagtttcaac catcgatcag aacttattga tgtacttatg tagctaggca cggtggcgcg
7921	tgcctgtaat cccagctact tggaagggtt aaggcaggag aatcgcttga acctgggagg
7981	cagaggttac agtgagtcaa gatcatacca ttgcactcca gtctgggcaa cagaatgaga
8041	ctctgtctca aaaacaaaaa acaaaccctt gtatgtgatt ttcctggata gcatctgtta
8101	catcttcaca aagataaaaa gtcagacttg gctgggcatg gtggctcaca cctgtaatcc
8161	cagcactgag aggctgaggc aggcagatca cttgaggtca ggaatttgag accaggctgg
8221	gcagcatggt gaaaccccgt ctctacaaaa aatacaaaaa ttagccgggt gtggtgtcac
8281	gcacctgtat tcccaagcta ctcaggaagc taaggcagga gaatcacttg aacccagagg
8341	tggaggtttg cagtgagttg agattgtgcc attgcactcc agcctgggcg acagagtgag
8401	actctgtgtc aaaaataaaa taaaataaaa ttttaaaaaa ggcagatttt tttttcttct
8461	tggtattgtt accttattat agtaataata agtgcatagt gcatgctgag ataagcaatc
8521	ataatttgtt attgcggccg ggcatggtgg ctccagccta taatcccagc actttggtca
8581	ggagttcaag gccagcctgg ccaatatagt gaaactccat ctctactaaa atacaagaaa
8641	ttacctgggc atggtggcag ttgctggtga tccccagcta cttgggaggc tgaggcagga
8701	gaatcgcttg aacctgggaa gcagaggttg cagtgagcca agattgcacc actgcactcc
8761	agcctgggtg acagagtgag actctgtctg aaaataataa taataataat ttgttattgc
8821	ttttattgcc ttagtttaca tagggaatca aagtttatac tttgatttat aaaagttgct
8881	ttgattctag ttcacagaac cagaatcttt catataaagg tattagaggg cccagtgtgg
8941	tggctcatgc ctgtaatccc agcatattgg gaggctgagg agggaggatc actttaggag
9001	tttgaggcca gcctaggcaa catagtgaga ccttgtctct acaaaaaatt ccaacattag
9061	ctgggcatgg tggcatgtgc ctgtagtccc atttatttgg ggggctgagg caggaggatc
9121	acttgagccc acgaggttca atccaggttg cagtaagcca tgatcctgcc actgcactcc
9181	agtttgggta acagagcgaa gctatgtctc aaaaaaagaa aaaaaaagta ttctaaatcc
9241	aaatttaata tataaaacta aatgcaggcc aagtgtggtg gcatatacct ataatcacaa
9301	cactttggga ggctgaggtg ggaggattgc ttgagcccaa gagttcaaga ccagcctagg
9361	taacacagta agaccccatc tctacaaaaa gtagaaaaat tagcctggca tggtggtgag
9421	tgcttttaat cccaactact tagggggctg agatgggaag attgcttgag cctcagagtt
9481	tgaggctgca gtgggccgtg atcgctccac tgatcgctct aaagtgagac cctgtctcaa
9541	aaaaaaagaa aatagaagaa aactaaatac attcaataag actttgatct cttttccaag
9601	gtgtaaatat attttgggaa attttccagt tactttgttc tcattttaat gtaataatct
9661	aagtcttggt tttctaagga aaagttttct cttattatat cttttgttaa tgtttctctc
9721	ccatttcttt tgatctgatc ttcagataca tgattatctt cactgctaaa tttgtgttct
9781	ctggcctcta catttataat ttctcataat tctttatcta agtatttctt ccctacctac
9841	tgaagaaaac tcaagttttc ttccacctta atgattatgc tgtgtctgtg agttttcttc
9901	atgactcttt acagtacaag ttttttgttt ttgttttttt aatggtcaga tggatagaac
9961	aacacaggtt ttgtttgttt tgttttaact tttaaaaaaa ttataataga taaagggtct
10021	cactacgttg tccaggctga tctcatactc ctgggctcaa gcaatccacc cacctctgcc
10081	tcccaaagtg ctgggattac agtcatgagc caacatgcct gggcagtaca ggtttttttt
10141	gagacggagt tttgttcttg ttgccgaggc tggagtgcaa tggcacaatc ttggctcacc
10201	acaaagtctg cctcccaggt tcaagtgatt ctcctgcctc agcctcctga gtagctggga
10261	ttacaggcat gtgccaccac gcccagctaa ttttgtattt ttagtagaga cggggtttca
10321	ccatgttggc caggctggtt tcgaactgct gacctcaggt gatctgccca cctcggcctc
10381	ccaaagtgct gggattacag gcatgagcca ccatgcccag ctgtagtaca ggttttaata
10441	tgctaaatac tcttcctttc tttattaatg tgcatggaag ttctaatatt tttttcccat
10501	accccagaga gtccatattt tggaatcaac aacactagcc tttgttgaca agtgtctctc
10561	ttgggttcct tctttgtgtc ctccactgaa ttttggggtt cataaaattt catttgttgt
10621	gcttgcttaa ttccctggga atcagactgt tcctgatcgg atgacatttc tggttaattc
10681	tttagttggc aggaaataga cacaggaaac gtggtcagtt tctgattctg gcgttgagag
10741	accctttctc cttttcctct ctctcagtgg gcgacagatg cgaaagaaac gagttccagt
10801	gccaagacgg gaaatgcatc tcctacaagt gggtctgcga tggcagcgct gagtgccagg
10861	atggctctga tgagtcccag gagacgtgct gtgagtcccc tttgggcatg atatgcattt
10921	atttttgtaa tagagacagg gtctcgccat gttggccagg ctggtcttga atttctggtc
10981	tcaagtgatc cgctggcctc ggcctcccaa agtgctggga ttacaggcac cacgcctggc
11041	ctgtgacacg attcttaacc cctttttgat gatggcggct ggaaaagtgg ccagtggatt
11101	ttgatgtatt caatcatgaa ttaggaggtg gggagagaat gaattattgg agctttcctt
11161	aaagccatta aatggctcta ttgttttttc aattgatgtg aatttcacat aacatgaaat
11221	taaccagctc agtggcatta atacatctgc aatgctgtgt ggccaccacc tctatcttgt
11281	tccaaaactt tgcataacct aatgtctttt tttttttttt tttttgagac ggagtctcgt
11341	tccatcaccc aggctggagt gcagtggtgt gatctcagct cactgcaacc tccgcctccc
11401	aggttcacgc catcctcctg cctcagcctc ccgagtagct gggactacag gcaccctcca
11461	ccacatccgg ctaatttttt gtatctttag tagagatggg gtttcaccat gttagccggg
11521	atggtctcga tctcctgacc tcgtgatcca cctgcctccg cctcccaaag tgctggcatt
11581	acaggcgtga gccaccatgc ccggcctatt ttttttttta agagatggag tctaattctg
11641	ttgcccaggc tggagtccag tggtaccatc atacttcact gcagccttga cctcttgggc
11701	tcaagtgatt ctcttgcctc gaactcccaa agtattggga ttacaggtgt gagccaccgc
11761	actcagccta atgtccagtt tttaacaagc tccatttaaa tgccctccgt tttgacccat
11821	aaaggggtag gcttggccgg gcacaatggc ttgtgtctgt agtcccagct acttgggagg
11881	ctgaggcaga aaggcagaaa gattgcttta taaagcccag gagtttgagg gccacctggg
11941	tggcatagct agacctcatc tctaaaaaat aagtaataaa taaatatttg tttttgtttt
12001	tttctttttc ttttcttttt tttttttttt tgagacggag tcttgctctg ttgcccaggc
12061	tggagtgcag tggcgcgatc tcagctcact gcaagctgtg cctcctgggt tcatgccatt
12121	ctcctgcctc agcctcccga gtagctggga ctacaggcgc ccactaccac gcccagctaa
12181	ttttttgtat ttttagtaga gatggggttt caccacgtta gccaggatgg tctcaatctc
12241	ctgacctcgt gatccgccag ctttggcctc ccaaagtgtt gggattacag gcgtgagcca
12301	ctgagcccgc cccatatgta tgtatatata tattttttta aaatgggaga ccaggcatgg
12361	tggctcatgc ctagaatccc agcactttgg gaagctgagg taggcggatc acttgaggcc
12421	atgagtttga gaccagcctg ctcaacatga tgaaacttct atctctacta aaaaaaaaag
12481	tgggattagg tcaggcacgg tggctcacac ctgtaatccc agcactttca gaggccgagg
12541	caggaggatc atgaggtcag gagatcgaga ccatcctggc taacacggtg aaaccccgtc
12601	tctactaaaa aaatacaaaa aattagccag gcgtggtggc gggtgcctgt agtcccagct
12661	actcaggagg ctgaggcagg agaatggcgt gaacccggga ggcggagctt gcagtgagcc
12721	aagatcgtgc cactgtactc cagcctgggc gacagagcaa gactctgtct caaaaaaaaa
12781	aaaaaaagtg ggattgacat tctcttcaaa gttctggggt tttcctttgc aaagacagga
12841	ttggcaaggc cagtgggtct tttttgtgtg tgtgtgtgtg acggagtctc actctgccac
12901	ccaggctgga gtgcaatggc aggatctcgg ctcaccgcaa cctcctcctc ccaggttaaa
12961	gtgattctcc tgcctcagcc tcccgagtag ctgggactac aggtgcccgc caccacaccc
13021	aactaatttt tgtattttta gtagagacag ggtttcacta tattggccag gctggtcttg
13081	aacccctgac ctcacgtgat ccacccgcct tggcctccca aagtgctggg attacaggcg
13141	tgagccactg tgctcggcct cagtgggtct ttcctttgag tgacagttca atcctgtctc
13201	ttctgtagtg tctgtcacct gcaaatccgg ggacttcagc tgtgggggcc gtgtcaaccg
13261	ctgcattcct cagttctgga ggtgcgatgg ccaagtggac tgcgacaacg gctcagacga
13321	gcaaggctgt cgtaagtgtg gccctgcctt tgctattgag cctatctgag tcctggggag
13381	tggtctgact ttgtctctac ggggtcctgc tcgagctgca aggcagctgc cccgaactgg
13441	gctccatctc ttgggggctc ataccaagcc tcttccgccc ttcaaatccc cccttgacca
13501	ggaggcatta caaagtgggg atggtgctac ctcttcgggt ttgtcacgca cagtcaggga
13561	ggctgtccct gccgagggct agccacctgg cacacacact ggcaagccgc tgtgattccc
13621	gctggtcgtg atccccgtga tcctgtgatc cccgccccgt gaggctgaac acatagtgac
13681	gcttgctagc caagcctcaa tgacccacgt aacatgaagg gggaaaagcc agaaagttct
13741	gccaaggagc aaggccaaga atcccgaagg gaaatggact ttgaagctgg gcgtcttctt
13801	ggctgtctta atacaagtgg cacatccaaa tccaaaaccc cgaaattcaa agtcttgagc
13861	acccgaaatt ctgaaacgtc ttgagcactg acctttagaa ggaaatgctt attggagcat
13921	tttggatttc ggatttttac cactgagtgt ggagtcctaa ttaggaaaaa aaccaggctg
13981	accgaaccaa aggaaagcaa taaaagaagg cagatagggt caggcacggt ggctcacccc
14041	tgtaatccca gccttttgag aggctgaggc gggtggatca cttgaggtca ggagttcgag
14101	agcagcctgg ccaacacggt gaaaccccat ctctactgaa aatacaaaaa ctagccaggt
14161	atggtggcgt ctgcctgtaa tcccagctac tcgggaggct gagacaggag aatcacttga
14221	acctgggagg cagaggttgc agtgagccaa tatcacgcca ttgcactcca gcctggggga
14281	caagagcgaa attctgtctc aaaaaaaaag aagaagaagg ccgacaaact atgtaactct
14341	gcctttctcc atggtccaga acacacagcc ctcctgcgta aataactcct tatcttcctg
14401	ctcccagcta tcatcagaca cctcggctga tagaaaattg caagttagct cactgcaacc
14461	tcggcattat aagtactgca caaagccctc ttcagcgcac agcacaagca ccattctata
14521	aaatctccag caagcggcca ggtgcagtgg ctcatacctg taatcccagc attttgggag
14581	actgaggcgg gcggatcacc tgaggtcagg agtttgagac cagcctggcc aacatggtga
14641	aaccccgtct ctattaaaaa tacaaaaaaa ttagccaggc gtggtggcag gtgcctgtaa
14701	tcccagctac ttggaaggct gaggcaggag aatcgcttga acccgggagg tggaagttgc
14761	agtgagccga gatcttgcca tcgcactcca gcctggggga caagagtgag acttcgtctc
14821	aaaaaaaaaa aaaaaaattc ccagcaagcc tttgtcttct ggcagtcagc tcctctcttg
14881	ctgacctgct cattgctttc ttgcaaggta ttttcctacc tactttctgg aataaatctg
14941	tctttctgta cttacaacta ccttttttaa aatttctttc ttttttgaga tggagtctca
15001	ctctgtttgc ccaggctgga gttcagtggt gcaatctcag ctcactgcaa cctctaccta
15061	ctgggttcaa gcgattctcc tgcctcagct tcccgagtag ctgggattac aggcgtgcac
15121	cagcacgcag gctaattttt gtatttttag tagagacggg gtttcaccat gttggccaag
15181	gtggtcttga actcctgacc tcaagtgatc ctcccacctc agcctcccaa agcgctagga
15241	ttacggccat gagccactga ggccggctgc acctacaact gtcttgataa attcttaccc
15301	ccacaccact ggtccagata gtcagtgctc acccacaaca ttaaggatat tccaaatttg
15361	aaacattcca aaatcagaaa aatattccaa ctctgaaaat attccaaaat ccaaaaaaat
15421	tcaaaatcca aaacacttct ggtcccaagc attttagaga agggatactc aacccaaaat
15481	aaggacagca attctataaa ttgtgctacc atcttgcagg tctcagttta acagctttac
15541	acctattagc gcaccagtgc tcatagcagt gctgggaaat gtgtacagat gaggaaactg
15601	aggcaccgag agggcagtgg ttcagagtcc atggcccctg actgctcccc agcccgcctt
15661	tccaggggcc tggcctcact gcggcagcgt ccccggctat agaatgggct ggtgttggga
15721	gacttcacac ggtgatggtg gtctcggccc atccatccct gcagccccca agacgtgctc
15781	ccaggacgag tttcgctgcc acgatgggaa gtgcatctct cggcagttcg tctgtgactc
15841	agaccgggac tgcttggacg gctcagacga ggcctcctgc ccggtgctca cctgtggtcc
15901	cgccagcttc cagtgcaaca gctccacctg catcccccag ctgtgggcct gcgacaacga
15961	ccccgactgc gaagatggct cggatgagtg gccgcagcgc tgtaggggtc tttacgtgtt
16021	ccaaggggac agtagcccct gctcggcctt cgagttccac tgcctaagtg gcgagtgcat
16081	ccactccagc tggcgctgtg atggtggccc cgactgcaag gacaaatctg acgaggaaaa
16141	ctgcggtatg ggcggggcca gggtgggggc ggggcgtcct atcacctgtc cctgggctcc
16201	cccaggtgtg ggacatgcag tgatttaggt gccgaagtgg atttccaaca acatgccaag
16261	aaagtattcc catttcatgt ttgtttcttt tttttctttt ctttctttat tttgtttttg
16321	agatggagtc tcactctgtg atttttttca tctctaaatt tcctacatcc atatggccac
16381	catgaggccc caggctggcc gatggttgct gttagcttat tgggaaatca ctgtttggaa
16441	ggtgctggtt gttttttgtt gtttgttgtt tttgtttttg tttttgtttt gagacggagt
16501	ctcgctctgt cgccagggtg gagtgcagtg gcgcgatcag ctcactgcaa cctccgcttc
16561	ctgggttcaa gccattctcc tgcctcagcc tcccaagtag cgcggattac aggcatgtgc
16621	caccacctcc ggctattttt ttttctattt agtagagatg gggtttcacc atgttagtca
16681	ggctggtcat gaactcttga cctcaggtga tccacccgcc tcggcctccc aaagtgctgg
16741	gattacaggc gtgcactgct gcacccagcc tttttttgtt tttttgagac agggtcttgc
16801	tgtcacccag gttgaagtaa ggtggcacga ttatggctca ctgcggcctt gatctccttg
16861	gctcaagcga tcctctcact tcagcctctc aagcagttgg aaccacaggc tgtaccacca
16921	agcctggcca atttttttgt acagacacag gctggtcttg aactcctggg ctcaagcaat
16981	cctcctgcct tggcctccca aagtgctggg attccaggca tgagccgctg cacccggcaa
17041	aaggccctgc ttctttttct ctggttgtct cttcttgaga aaatcaacac actctgtcct
17101	gttttccagc tgtggccacc tgtcgccctg acgaattcca gtgctctgat ggaaactgca
17161	tccatggcag ccggcagtgt gaccgggaat atgactgcaa ggacatgagc gatgaagttg
17221	gctgcgttaa tggtgagcgc tggccatctg gttttccatc ccccattctc tgtgccttgc
17281	tgcttgcaaa tgatttgtga agccagaggg cgcttccctg gtcagctctg caccagctgt
17341	gcgtctgtgg gcaagtgact tgacttctca gagcctcact tccttttgtt ttgagacgga
17401	gtctcgctct gacacccagg ctggagtgct gtggcacaat cacagctcac ggcagcctct
17461	gcctctgatg tccagtgatt ctcctgcctc agcctcccga gtagctgaga ttaaaggcgt
17521	ataccaccac gcccggctaa ttttttgtat ttttattaga gacagggttt ctccatgttg
17581	gccaggctgg tcttgaactc ctggtctcag gtgatccacc cgcctcggcc tcccaaagtg
17641	ctaggattac aggtgtgagc cactgcgcca ggcctaattt ttttgtattt ttagtagaga
17701	tgcggttttg ccatattgcc caggctggtc tcgaactcct gggctcaagc gatctgcctg
17761	ccttggcctc ccaaagtgct gggattacag gcacaaacca ccgtgcccga cgcgttttct
17821	taatgaatcc atttgcatgc gttcttatgt gaataaacta ttatatgaat gagtgccaag
17881	caaactgagg ctcagacaca cctgaccttc ctccttcctc tctctggctc tcacagtgac
17941	actctgcgag ggacccaaca agttcaagtg tcacagcggc gaatgcatca ccctggacaa
18001	agtctgcaac atggctagag actgccggga ctggtcagat gaacccatca aagagtgcgg
18061	tgagtctcgg tgcaggcggc ttgcagagtt tgtggggagc caggaaaggg actgagacat
18121	gagtgctgta gggttttggg aactccactc tgcccaccct gtgcaaaggg ctcctttttt
18181	cattttgaga cagtctcgca cggtcgccca ggctggagcg caatggcgcg atctcggctc
18241	actgcaacct ctgcctccca ggttcaagtg attctcctgc ctcagcctcc tgagtagctg
18301	ggattacagg cgcccaccac caagcccggg taattttttg tatgtttagt agagatgggg
18361	tttcactatg ttggccaggc tggtgttgaa ctcctgacct catgatccgc ccacctcggc
18421	ctcccaaagt gctgggatta caggcgtgac ccaccccatg aaaaaaaatt aaaaaatgaa
18481	gcgatgctgg gcgcggtgga tcacgcctgt aatcccagca ctttgggaag ctgaggcagg
18541	cagatcacga gggcaggaga ttgagaccat cctggctaat acggtgaaac cccatctcta
18601	ctaaaactac aaaaaattag ccgggtgtgg tggcaggcac ctgtgatccc agctactcag
18661	gaggctgagg caggagaatc gcttgaaccc aggaggtgga ggttgcagtg agccgggatc
18721	acaccattgc actccagcct gggtgacaga gtgagactct gtctcaaaaa aaaaaaaaaa
18781	aaaaaaagcg aattctgaaa tacatgaatt cttttcctta gatgcctgct tctgtcttga
18841	ggtttgttgt tgttatttcg aaacagagtc ttgctctgtc gctcaggctg gagtgcagtg
18901	gcatgatctt ggctcaccac aacctccggc tcccaggttc aagcgattct tctgcctcag
18961	cctcctgagt agctgggatt acagctgaat gccaccttgc tgggctaatt tttgtatttt
19021	tagtagagat ggggtttcac catgttggcc aggctggcct cgaactcctg acctcgagtg
19081	atctgcccgc ctcctgaagt gctgggatta caggcgtgag ccacctcgtc ctggtgaggg
19141	tttttttttt tccccaaccc tctgtggtgg atactgaaag accatattag gataactgta
19201	cagtatagag aaggcagtgg caagttttct ctgtcatata ccagagtggg cttgggcatg
19261	gtggcatact cctgtagtct cagctaatca ggaggctgag gaaggaggat cgcttgggcc
19321	caggagttgg agactgtagt gagctgtgat cacaccacca cacttcaatc tgggcaacag
19381	agcaagagac cctatctcta aaaaaaagta agtatttcgg acactgtggg ccatacggtc
19441	tctggtgcag tttctcaaca tggctgttgg gtgaacacaa ccacgcacag aacgcaaacc
19501	aatacacgtg gctgtgggcc cagaaaatgt tatttatgga cacaaaaatt ggaatttcat
19561	ataactgttt tgtgtcatga aaatgatttc cctttttatt tttatttttc ttctcaagta
19621	tttaaatatg taaaagccat ttttaggcct ggcaggatgg ttcacagctg taatcccagc
19681	actttgggag gtcgaggcgg gaggatcacg aggtcaggag atcgagacca tcctggccaa
19741	cacagtgaaa ccccgtctct actaaaaata caaaaaatta accaggcttg gtggcgcgcg
19801	tctgtagtcc cagctgctca ggaggctgag gcaggagaat cgcttgaatg caggaggcgg
19861	aggttgtagt gagccgaggt tgcaccactg cactccagcc tgagcgacag agtgagagtc
19921	cgcctcaaac aaaaaaatgt ttgcccatgc tggtcttgaa ctcctgggct caagctatct
19981	gcctgccttg gtctcccaaa gttctgggat tacaggcatg agctacagcg cccggacttt
20041	tgttgtttta tatctatata tctatatata acttgtttta tgtatatata taacttgttt
20101	tatatatata cataaactgc agtaaaaaac atgtaacata aaatttacct tctcaaacct
20161	tattaagtgc acagttctgt gccattagca aattcacact gttgtacaac atcacaacca
20221	ccatctccag aacttttttt ttttttttta ttctttttga gacagagtct cactcgtcgc
20281	acgggctgga gtgcagtggt gcgatctcgg ttcactgcaa cctccaccta ccaggttcaa
20341	gcaattctcc tgcctcagcc ccctcagtag ctgggattac aggtgcccgt cctaccacgc
20401	ccagctaatt tttgtatttt cagtagagac tgactgggtt tcaccatgtt ggccaggctg
20461	gtctcgaact cctgacctca agtgatcctc ccacctcagc ctcccaaagt gctgggaata
20521	caggcatgag ccactgcgcc cggccccaga actcttttat cttcccaaac tgaagctctg
20581	tccccatgaa acactcactc tccatcccct ccccaactcc tggcacccac cattctactt
20641	tctgtcccta tgaatgtgat ggctctaggg acctcctctg agtggaatca gacagcattt
20701	tccttttttg actggcttat ttcactgagc caagtgcggt ggcacacgcc tgtaatccca
20761	aaactttggg agaccgaggc gggcgcatca cctgaggtca ggagttcgag accagcccgg
20821	ccaacatggt gaaaccccat ctctagtaaa aatacaaaaa attagcctgt catggtcgtg
20881	ggtgcctgta atcccagcta agtgggaggc tgaggcagga gaatcgcttg tacccaggag
20941	gcggaggtcg cagtgagccg agatcgtgcc attacactcc agcctgggca acaagagtga
21001	aactccgtct ctcctaaaaa tacaaaaaaa ttagctgggc atggtggcac atgcctgtag
21061	tcccagctac ttgggaggct gaggcaggag aatcacttga acccgggagg tggaggttgt
21121	aatgagccaa ggttggcggc gaagggatgg gtaggggccc gagagtgacc agtctgcatc
21181	ccctggccct gcgcagggac caacgaatgc ttggacaaca acggcggctg ttcccacgtc
21241	tgcaatgacc ttaagatcgg ctacgagtgc ctgtgccccg acggcttcca gctggtggcc
21301	cagcgaagat gcgaaggtga tttccgggtg ggactgagcc ctgggccccc tctgcgcttc
21361	ctgacatggc aaccaaaccc ctcatgcctc agtttcccca tctgttaagt gtgcttgaaa
21421	gcagttagga gggtttcatg agattccacc tgcatggaaa actatcattg gctggccaga
21481	gtttcttgcc tctggggatt agtaattaag aaatttcagg ccgggtgcgt aatccctgta
21541	atcccaacac cttgggacgc cgaggcgggc agatcacctg aggtcgggag ttccagacca
21601	gcctgaccaa catggagaaa ccccgtctct actaaaaata caaaattagc cgggcttggt
21661	ggtgcatgcc tataatccca gctactcagg aggctgaggc aggagaatca cttgaacctg
21721	ggaggtggag gttgtggtga gccaagatcg tgccattgca ctccagcctg ggcaacaaga
21781	gtgaaactcc atccaaaaaa aaaagaaaag aaaagaaaaa aaagaaaaga aatttcagct
21841	gacacagctt cacactcttg gttgggttcc cgtggtgaat gatgaggtca ggtgatgact
21901	ggggatgaca cctggctgtt tccttgatta catctcccga gaggctgggc tgtctcctgg
21961	ctgccttcga aggtgtgggt tttggcctgg gccccatcgc tccgtctcta gccattgggg
22021	aagagcctcc ccaccaagcc tctttctctc tcttccagat atcgatgagt gtcaggatcc
22081	cgacacctgc agccagctct gcgtgaacct ggagggtggc tacaagtgcc agtgtgagga
22141	aggcttccag ctggaccccc acacgaaggc ctgcaaggct gtgggtgagc acgggaaggc
22201	ggcgggtggg ggcggcctca ccccttgcag gcagcagtgg tgggggagtt tcatcctctg
22261	aactttgcac agactcatat cccctgaccg ggaggctgtt tgctcctgag ggctctggca
22321	ggggagtctg ccgccctgtt aggacttggg cttgccaggg ggatgcctgc atatgtccta
22381	gtttttggga atatccagtt aacggaaccc tcagccctac tggtggaaca ggaaccggct
22441	ttcctttcag ggacaacctg gggagtgact tcaaggggtt aaagaaaaaa aattagctgg
22501	gcatggtgcc acacacctgt ggtcccagct actcagaagg ctgaggcggg aggattgctt
22561	gagggcagga ggattggttg atcctcccac ctcagcctcc ggagtagctg ggacctcagg
22621	tgcatgccac tatgcctggc taattttctt ttttcttttt tttttttttt cgagacggag
22681	tctcgctctg ttgcccaggc tggagtgcag tggcaggatc tcggctcact gcaagctccg
22741	cctcccgggt tcacgccatt ctcctgcctc agcctcccca gtagctggga ctacaggagc
22801	ccgccactgc accaggccaa tttttttgta tttttagtag agacggggtt tcactgtgtt
22861	agccaggatg gtctcgatct cctgacttcg tgatccgccc acctcggcct tccaaagtgc
22921	tcggattaca ggcgtgagcc actgcgccca gccgctaatt ttcatatttt tagtaaaaac
22981	agggtttcac catgttggcc aggctagtct tgaactcctg aacccaagtg atcctcctgc
23041	cttggcctcc caaagtgctg ggattacaga caccacacct ggctattatt attttttaga
23101	gacagggtgc tgctctatct tccagcctgt agtgcagtgc agcctccatc atagctcgct
23161	gcagccttga cctcctgggt tcacgtgatc gtcccgccta agcctctgga ggagctggga
23221	gtactggcat gtgccaccat gcctggttaa tttttttttt tttttttttg agacagagtc
23281	tcattctgtc acccaggctg gagtgcggtg gtgcgatctt ggcttactga aacctccacc
23341	tcccaggttc cagcaattct cctgcctcac ccttctgagt agctgggatt acaggttccg
23401	gctaccaaac ctggctagtt tttgtatgtt tagtagagac agggtttcac catgttggtg
23461	aggctggtct cgattctccc gcctcagcct cccaaagtgc tgggattaca ggcttgagcc
23521	accgtgcctg gctttttttt tttttttttt ttttgtggca ataaggtctc attgtcttgc
23581	ccaggctagc cttatgctcc tagcctcaag tgatcctcct ccctcagcct cccaaagtgc
23641	tgggattaca ggtgggcgcc actgtgcctg ttcccgttgg gaggtctttt ccaccctctt
23701	tttctgggtg cctcctctgg ctcagccgca ccctgcagga tgacacaagg ggatggggag
23761	gcactcttgg ttccatcgac gggtcccctc tgaccccctg acctcgctcc ccggaccccc
23821	aggctccatc gcctacctct tcttcaccaa ccggcacgag gtcaggaaga tgacgctgga
23881	ccggagcgag tacaccagcc tcatccccaa cctgaggaac gtggtcgctc tggacacgga
23941	ggtggccagc aatagaatct actggtctga cctgtcccag agaatgatct gcaggtgagc
24001	gtcgcccctg cctgcagcct tggcccgcag gtgagatgag ggctcctggc gctgatgccc
24061	ttctctcctc ctgcctcagc acccagcttg acagagccca cggcgtctct tcctatgaca
24121	ccgtcatcag cagagacatc caggcccccg acgggctggc tgtggactgg atccacagca
24181	acatctactg gaccgactct gtcctgggca ctgtctctgt tgcggatacc aagggcgtga
24241	agaggaaaac gttattcagg gagaacggct ccaagccaag ggccatcgtg gtggatcctg
24301	ttcatgggtg cgtatccacg acgctgaggg ctgcagaggg aatggaggga gcaggaagga
24361	gcttcaggaa ctggttagtg ggctgggcat ggtggctcaa agcacctgta atcccagcac
24421	tttgggaggc caaggtgggt ggatcatcaa gaccagcctg accaacatgg tgaaacctcg
24481	tctctactaa aaatacaaaa attagccggg tgtggtggtg ggcacctgta atcccagctg
24541	ctcgggaggc tgaggcagga gaatcacttg aacctgggag atggaggttg cagtgagcca
24601	agacagcccc actgcactcc agcctgggtg acagagtgag actccgtctc aaaaaaaaaa
24661	aaaaaaacta aacaaaaaac tggttagtgg ctagacaaca ggatggtatc ttccaagccc
24721	atggctgact cagcagctcc tgggtcaaga cactgtgacc tgtgtcccct ggcaggaagc
24781	atcgcccctg ccacctgccc ggtgtactct gtacctgtca ggtgacatct gctacctaag
24841	cacgtgagag gtggcatttc acagtttcag tgtggtgctg acaacccggg acgcacactg
24901	tccttgcagc tacaatcagg aggtgaatgt tgggtttcca gcagagaaca ctggagaagg
24961	cacacttggt gtctggaagg gaaaagcagg gaagagagca tcatcagatg cctgcgggtg
25021	aaggtgggcc cgctatggcc agcgtccctt tttattttta tttatttatt tatttgagat
25081	ggaatctcgc tctgtcgccc agactgtagt gcagtggtgc gatcacggct cactgcaagc
25141	tccgcctcac aggttcacgc cattctcctg cctcagcctc ccgagtagct gggactacag
25201	gcacccgcca ccacgcccgg ttaatttttt gcatttttat tagagacggg gtttcaccgc
25261	gttagccagg atggtctaaa tctcctgacc ctgtgatcca cccgcctcgg cctccctaag
25321	tgcttggatt acaagcgtga gccaccacgc ccggccccct ttttattttt tattttttga
25381	gacggagtct cgctctgtcg cccaggctag attgcagtgg cgtgatctcg gctcactgca
25441	gcctccgcct cccaggttca agtgattctc ctgcctcaac ctcccaacta attaggatta
25501	caagcatgta ccaccatgcc tgactaattt tttgtatttt tagtagagac tgggtttcac
25561	catgttggct aggctggtct cgaaccctta gcctcaagta atctgcctgc ctcagcctcc
25621	caaacagcgg ggattacagg catgagccac tgtgcccaac ccaaccctgg atctctttta
25681	aacaagacaa tgctcgctgt tgccacagaa caatgggtgg ggtacatgtg gcccagtgtg
25741	tttggccaca taactgccag gccagaggga aagagactct cagactgtct ccactcagat
25801	acaaatgtgt gtgttgtgtg cgtgtgttct ggtctcatat ttgtttgttt tgagacaggg
25861	tgtcgctctg tcactgagtc tggagtgcag tggcgcaatc agagttcact gcagcctcaa
25921	actcttgggc tcagttgatt ctcccacttc agcctcccaa gtagctggaa ctacaggtga
25981	acaccactgt gcccagctaa tttattttat ttttagtaga gatgaggtct cactatgttg
26041	cccaggctgg tcttgacctc ctagcctcaa gcaatcctcc tgccttggtc tcccaaagtg
26101	ctgggattac acgtgcgagc cattgcgcat ggcttgtgtt cttgtgtttc ttcctttttc
26161	tttcgagatg gcgtctcagt ctgccaccca ggctggagtg cagtggtgtg atcatagctc
26221	actgtagcct caacttcctg ggctcaagca atcctcttga tttcagcctc ccgggcctgg
26281	ccagcatggt gaaaccccgt ctctactaaa aatacaaaaa tgtagccagg cgtggtggtg
26341	ggcgcctgta atcccagcta caccagaggc tgaggcagga gaatcgcttg agcctggaag
26401	gtggaggttg cagcaagcca agatcgtgcc actgcactcc agcctgggca acagagacag
26461	actctgtctc aaaaaaaaaa aaaaaaaacc caaacaagcc acatttggag tttggggttc
26521	ccagcaggac tatttcccaa gcctgagcct ggctgtttct tccagaattc gttgcacgca
26581	ttggctggga tcctcccccg ccctccagcc tcacagctat tctctgtcct cccaccagct
26641	tcatgtactg gactgactgg ggaactcccg ccaagatcaa gaaagggggc ctgaatggtg
26701	tggacatcta ctcgctggtg actgaaaaca ttcagtggcc caatggcatc accctaggta
26761	tgttcgcagg acagccgtcc cagccagggc cgggcacagg ctggaggaca gacgggggtt
26821	gccaggtggc tctgggacaa gcccaagctg ctccctgaag gtttccctct ttcttttctt
26881	tgttttttct ttttttgaga tgaggtcttg gtctgtcacc caggctggag tgcactggcg
26941	caatcgtagc tcactgcagc ctccacctcc caggctcaag tgatcctcct gcctcaccct
27001	cctgagtagc tgagattaca gacacgtgcc accacggcag actaatttta ttttattttt
27061	gggaagagac aaagtcttgt tatgttggcc tggctggtct caaactcagg gtgcaagcga
27121	tcctcccgcc tcagccttcc aaactgctgg gattacaggc gtgggccacc gtacccagcc
27181	tccttgaagt ttttctgacc tgcaactccc ctacctgccc attggagagg gcgtcacagg
27241	ggaggggttc aggctcacat gtggttggag ctgcctctcc aggtgctttt ctgctaggtc
27301	cctggcaggg ggtcttcctg cccggagcag cgtggccagg ccctcaggac cctctgggac
27361	tggcatcagc acgtgacctc tccttatcca cttgtgtgtc tagatctcct cagtggccgc
27421	ctctactggg ttgactccaa acttcactcc atctcaagca tcgatgtcaa cgggggcaac
27481	cggaagacca tcttggagga tgaaaagagg ctggcccacc ccttctcctt ggccgtcttt
27541	gaggtgtggc ttacgtacga gatgcaagca cttaggtggc ggatagacac agactataga
27601	tcactcaagc caagatgaac gcagaaaact ggttgtgact aggaggaggt cttagacctg
27661	agttatttct attttcttct ttcttttttt tttttttttt gagacagagt tttgctctcg
27721	tttcccaggc tggagggcaa tggcatgatc tcggctcacc gcaacctcca cctcccaggt
27781	tcaagtgatt ctcctgtctc aggctcccca gtagctggga ttacaggcat gcaccaccac
27841	catgcccggc taattttgta tttttagtag agacggagtt tctccatgtt ggtcaggctg
27901	gtctcgaact cccgacctca ggtgatctgc ctgcctcggc ctcccaaagt gctgggatta
27961	cagacttgag ccaccgcgcc cagctatttc tgttttcttt ctttcttctt cttctttttt
28021	tttttctaag agacaggatc tcactctgtc cccaggcagg agtgcagtgc tgtgatcata
28081	gctcactgca gccttaacct cctgggctca agtgatcttc ccacctcagc ctcccaagta
28141	gctggaacta caggtgcaca ccaccatgcc cagctcattt ttgtattttt tttttttttg
28201	agacagtctc gttctgtcac cccggctgga gtgcagtggt acaatcttgg ctcactgcaa
28261	cctctgcctc ccaggttcaa gcgattctcc tgcctcagcc tcctgagtag ttgagattac
28321	aggcatgtgt gccatcatac ctggctgatt tttgtatttt tttttagaga tggggtctca
28381	gtatgttgac caggcttgtc ttaaactccc ggcctcaagt gatcctccca cttcagtctc
28441	ccaaagtgct gggattacag gcatgagcca ctgcggccgg tttgttttct tttttttttc
28501	gttttttgga gacggaattt cacctttgtt gcccaggatg gagtgcaatg gcacgatatc
28561	gcctcaccac aacctctgcc tcctgggttc aaaccatttt cctgcctcag ccttcttagt
28621	agctgggatt acaagcatgt gccaccacgc ccggctgatt ttgtattttt agtagagatg
28681	gggtttctcc atgttggcca ggctggtctc gaactcctga cctcaggtca ttcgcccacc
28741	tctgcctccc aaagtgctgg gattacaggc gtgagccacc gtgcccggtg gtttgtattc
28801	tttttactga gagtcgtgaa aggcagtgat cctctgtcac atgtgatctt ggctctcagg
28861	ggacatttgg caatttctag agattttttg gttgtcacaa gtcaatgggg aagactgttg
28921	gcatttagtg ggtagaggct ggtgacgctg ctgaacaccc agaacaggga agtagcaggc
28981	cctagataga gccatcgtgg ggaaaccctg ctctaaggaa atggcgctat tttataaccc
29041	cacgttcctg gcatgattac caacagccaa aagtggagtc cccccaagtg tgttcgtcca
29101	tttgcattgc agtaaaggaa tagctgaggc cgggtaattt ataaagaaaa gagatttaaa
29161	ctgggtatgg cagtttatgc ctataatccc agaactttgg gaggctgagg caggaggatc
29221	gcttgagtcc aggagtgtga gaccgagacc agcctggcca acatgacgaa actctgtctc
29281	tacaaaaaat acaaaaagta ggccaggcac ggtggttcac gcctgtaatc ccagcacttt
29341	gggaggccga ggcgggcgga tcacgaggtc aggagatcga gaccatcctg gctaacacgg
29401	tgaaaccccg tctctactaa aaatacaaaa acaaaattag ccgggtgtgg tggcaggcgc
29461	ctgtagtccc agctactcgg gaggctgagg cgggagaatg gcgtgaaccc gggaggcgga
29521	gcttgcagtg agccaagatc gcgccactgc actccagcct gggtgaccga gttgagactc
29581	cgtctcaaaa aaaaaaaaaa aaaaaaaaat acaaaaagta gccaggtgtg gtggcaggca
29641	cctgtaatcc tgggttctcg agaccgaggc atgagaattg cctgacccca ggaggtggag
29701	gctgcagtga gccaagatca tgccactgca ctccagcctg ggcgacagag tgggactctg
29761	tctcaaaaaa caacaaaaaa aaagttctgg aaatggatgg tggtgatggt gatacttcca
29821	caacagcgtg aatctgctta aggccaccga actgtgcact cacaaatagt cgagatggta
29881	cattttatgt tatgtgtatt tcaccacaat taaaaactag ttgtgggcca ggtgtggtgg
29941	ttcatgcctg taatcccagc actttgggag gtcagaggga ggtggatcat gaggtcagca
30001	gttcgagacc agccaggcca acatggtgaa accccatctc tactaaaaat acaaaaatta
30061	gccaggcgtg gtggcacatg cctgtagtcc cagctacttg agaggctgaa gcaggagaat
30121	cgcttgaacc tgggaggcta agattgcagt gagccgagat cgtgccactg cactccagcc
30181	tggacgacag agtgagactt cgtctcaaaa aaaaaaccaa aaaaaaaatt agctgtgggt
30241	caggcactgt ggctcacgcc tgtaatccca gcactttggg agaccgaggt aggtggatgg
30301	cctgaggtca ggagttcgaa tccagcctgg ccaacatggt gaaagcccgt ctctactaaa
30361	aatacaaaaa attagtcagg tatgttggca cacctgtaat cccagctact cgggaggctg
30421	aagcaagaga atcgtttgaa cccaggaggt ggacgttgca gtgagccgag attgggccac
30481	tgtactccag cctgggcaac aaaagtgaaa ctctgtctga aacaaacaaa caaacaaaca
30541	aacagacaaa caaaaaaact agttgtggag agagggtggc ctgtgtctca tcccagtgtt
30601	taacgggatt tgtcatcttc cttgctgcct gtttaggaca aagtattttg gacagatatc
30661	atcaacgaag ccattttcag tgccaaccgc ctcacaggtt ccgatgtcaa cttgttggct
30721	gaaaacctac tgtccccaga ggatatggtt ctcttccaca acctcaccca gccaagaggt
30781	aagggtgggt cagccccacc cccccaacct tgaaacctcc ttgtggaaac tctggaatgt
30841	tctggaaatt tctggaatct tctggtatag ctgatgatct cgttcctgcc ctgactccgc
30901	ttcttctgcc ccaggagtga actggtgtga gaggaccacc ctgagcaatg gcggctgcca
30961	gtatctgtgc ctccctgccc cgcagatcaa cccccactcg cccaagttta cctgcgcctg
31021	cccggacggc atgctgctgg ccagggacat gaggagctgc ctcacaggtg tggcacacgc
31081	cttgtttctg cgtcctgtgt cctccaactg ccccctcctg agcctctctc tgctcatctg
31141	tcaaatgggt acctcaaggt cgttgtaagg actcatgagt cgggataacc atacttttct
31201	tggatggaca catcagcacc gggcttgaca tttacccagt tcccctttga tgcctggttt
31261	cctctttccc ggccccctga agaggtgatc tgatttctga caggagccct gagggaggaa
31321	atggtcccct ttgttgactt ttctttttct ttattttttt cttttgagat ttgctgtcac
31381	ccagcctgga atgcagtggt gccatcttgg ctcactgcta cctctcccac tgggttcaag
31441	caattctcct gcctcagcct cccaagtagc tgggattaca agcatgcgcc accatgcctg
31501	gctaagtttt gtatttttag tacagacagg gtttctccat ggtggccagg ctggtcttga
31561	actcctgacc tcaggtgatc ctcccacctc tgcctcccga agtgctacga ttacaggcat
31621	gagccaccgc gcccatcccc ctttgttgac ttttctcatc ctctgagaaa gtctcagttg
31681	aggccagcac ctccctcaag tgaattgaat ctcccttttg aacaacaaca aataacaata
31741	tgacccagac gtggtggctc acacctgtgg tcccagctac tcgggaggct gaggtgtgag
31801	gattgcttga gcccaggagg tcaaggctac agagagctat aatcacacca cttcactcca
31861	gcctggggga caaagtgaaa ccctgtctga aaaaaacaaa aaaagaaaaa ggaaaaagaa
31921	acaatacgat cacaaagtag atattcatag tgtttatttt cagtactctt tttttttttt
31981	tttttttttt ttgagacgga gtcttgctct gttgcccagg ctggagtgca gtggcacgat
32041	cttggctcac tgcagcctct gcctcccagg ttcaagcgct tggctcactg caacctccgc
32101	ctcctgggtt caagcgcttc ttctgcctca gcctccccag tagctgggac tataggcacg
32161	tcccactacg cccagctaat tttttgtatt ttttagtaga gatggggttt cactatgtta
32221	gccaggatgg tctcgatctc ctgacctcgt gatctgcctg ccttgggctc ccaaagtgtt
32281	gggattatgg gcatgagcca ctgcacctgg cctttttttt tttttttttt gagatggagt
32341	ttcgctcttg ttgcccaggc tggagtgcaa tggtgtgatc tcggctcact gcaacctctg
32401	cctcctgggt tcaagcaatt ctcctgcctc agcctcccga gtagctggga ttacaggcac
32461	ctgccaccac gcctggctaa tttttgtact tttagtagag acggggtttc tccatgttgg
32521	tcaggctggt ctcaaactcc tgacctcagg tgatccaccc acctcggcct cccaaagttc
32581	tgggattaca gacatgagcc accgcgcctg gccgtgtctg gcctttttta gttatttctt
32641	tttttttttt tttttttttt gagacagagt cttactccgt cgcccaggct ggagtgcagc
32701	ggtgcgatgt ctgcgcactg caagctccgc cccctgggtt catgccattc tcctgcctca
32761	gccttctgag tagctgggac tgcaggcgcc tgccactacg cccggctact tttttgtata
32821	tttagtagag atggagtttc actgtgttag ccaggatggt ctcgatctcc tgactttgtg
32881	atccgcccgc ctcggcctcc caaagtgctg ggattacagg cgtgagccac catgccaggc
32941	tttttttttt tttttttttt ttgagacgga gtcttgctct gtcgcccagg ctggagtgca
33001	gtgccatgat ctcagctcac tgcaagctcc acttcccagg ctcacgccat tctccagcct
33061	cagcctccca agtagctgag actacagggg cccgccacca cactcggcta atttttttgt
33121	atttttagta gagacggggt ttcaccatgt tagccaggct ggtcttgaac tcctaacctc
33181	aggcgattca cctgcctcgg cctcccaaag tgctgggatt aaaggtatga gccacctcgc
33241	ctggtgtgag ccacctcgcc cagcctgagc cacctcaccc agcctaagcc actgtgcctg
33301	gcctgatttt ggacttttta aaaattttat taataattat ttttgggttt cttttttttg
33361	agacagggtc ttactctgtc atccaggcca tcctgtctgt ctgtcatccc agtgatggga
33421	tcataccttg ctgcagcctc tacctcctgg gctcaagcga tcctcccccc tcagcctcct
33481	gagtagctgg gagtacaggt gtgcaccacc acacctggct aatttttttt tttttttttg
33541	tatatagaga tggtattttg ccatgttgac caggctagtc ttaaactcct ggactcactc
33601	aagagatcct cctgccttgg cctcccaagg tcatttgaga ctttcgtcat taggcgcaca
33661	cctatgagaa gggcctgcag gcacgtggca ctcagaagac gtttatttat tctttcagag
33721	gctgaggctg cagtggccac ccaggagaca tccaccgtca ggctaaaggt cagctccaca
33781	gccgtaagga cacagcacac aaccacccga cctgttcccg acacctcccg gctgcctggg
33841	gccacccctg ggctcaccac ggtggagata gtgacaatgt ctcaccaagg taaagactgg
33901	gccctcccta ggcccctctt cacccagaga cgggtccctt cagtggccac gaacattttg
33961	gtcacgagat ggagtccagg tgtcgtcctc actcccttgc tgaccttctc tcacttgggc
34021	cgtgtgtctc tgggccctca gtttccctat ctgtaaagtg ggtctaataa cagttcttgc
34081	cctctttgca aggattaaat gggccaaatc atatgagggg ccaggtcctt caggctcctg
34141	gttcccaaag tcagccacgc accgtgtggg tcccaaaatt ttatcaaggc acattcgttg
34201	cctcagcttc aggcatctgc ccaaaaaggc caggactaag gcaaggagag ggagggattc
34261	ctcagtactc agcttttcac agaggctcca aaaggctaag gaatccagta acgttttaac
34321	acaattttac aatttttttt tttgagacgg agttttgctc ttgttgccca ggctggagtg
34381	cagtggcacg atctcggctc actgcaacct ctggctcccg ggttcaagcg attctcctgc
34441	ctcagtctcc cgagtagctg ggattacagg catgcgccac cacgctcggc taattttgta
34501	tttttagtac agaaggggct tctctgttgg tcaggctggt cgtgaactct caacctcagg
34561	tgagccaccc gcctgagcct cccaaagtgc tgggattaca ggtgtgagcc accacgcctg
34621	gccttttttt tgagacagag tctcgctctc gcccatgctg tactgcagtg acgcagtctg
34681	ggctcactgt aacctccgct tcccaggttc aagtgattct tctgccgcag cctcccatgt
34741	agagtagctg ggattacagg cacccgccac catgcctggc taattcttgc atttttagta
34801	gagatggggt ttcacagtgt tggccaggct ggtctcaaac ttctgacctc aagtcatctg
34861	cctgccttgg ccctgccaaa gtgctgggat tatagatgtg agccaccgcg cctggcctac
34921	agtttattct ttggtggctc acacctgtaa tctcagcact ttgggaggcc aaggtgggag
34981	aatggcttga gcccaggagt tcaagtccag cctgggcaac atagcaagac cctatctcta
35041	ctacaaaata aataataaat aaactaattt tttttctttt aaaacccaac tattcaacat
35101	ggcaatgcaa tatattaaaa aaattttttt tttctttgaa acggagtctc tcactgtcac
35161	ccgggctgga gtgcagtgtc gccatcttgg ctcactgcaa cctccgcctc ccaggtccaa
35221	gtgattctcc tgcttcagcc tcccgagtag ctgggattac aggcacccac caccataccc
35281	agctaatatt tttgtatttt tagtagagat ggggtttcac tatgttgggc aggctggtct
35341	ggaactcctg acctcgtgat ctgcccgagg atcggcggcc tcccaaagtg ctggggattg
35401	caggcatgag ccaccgtgcc cagccaaaac ttttttattt ttattttttt gggacacggt
35461	ctcactgtgt accccagact ggagtgatag agtgctgtca tggctcactg cagcctcaac
35521	ctccctgggc tcaggtgatc ttcctgcttc agtctcccag gtagctggga ctacaggcat
35581	gagccaccac acccagctaa tttttgaatt tttttgtaga gacagggttt caccttgtgg
35641	cccagacttg tctctaactc cagggctcaa gcgatctgcc caccttggcc tcccaaagtg
35701	ctgagattaa tgcaatttaa aaaatttttt ggccaggcct ggtggctcat gcctgtattc
35761	acaacacctt gggaggcaaa ggtgggcaga tcacttgagg tcaggagttc gagactagcc
35821	tggccaacat ggtgaaaccc cctgtctact aaaaaaatac aaaaattacc tgggcacagt
35881	ggtgggtgcc tgtaatccca gctacttggg atgctgaggg tggagaattg cttgaacctg
35941	ggaggcagaa gttgcagtaa gccaagatca tgccactgga ctccagcctc agtgacagag
36001	caaaactctg tctccaaaaa aattgttttt tttttttttt tttcaaatca tcacactaca
36061	gccaaggcct ggccacttac ttttgtaaat aaagttttat tggagccagt ggaccagtga
36121	ggccgaatct tgcaggtgta agatcacagt ctatccttga aaattttgat attttgttca
36181	ttgggtggtt tttcattaat ttaaatttta aaaaataaca tattaaaggc tggtgtggag
36241	gtgcacgcct gcagtcctag ctactcccag aggctgaggc gggagacttg cttgagccca
36301	agagttgaag tccagcctgg gcaacatagc gagaccccca tctctaaaaa taaaaataat
36361	gcattagaat attattggat tcctgggcag ggcacagtgg ctcacacctg taatcccagc
36421	actttgggag gctgaggtgg gtggatcacc tgaggtcagg agtttgagac cagcctggcc
36481	aacatggtga aaccccgtct ctactaaaaa tacaaaaatt agccaggcgt ggtggcaggt
36541	gcctgtaatc ccagctactc gggaggctga agcacgagaa tcgcttgaat ccaggaggcg
36601	gaggttgcag tgagctgaga ttgcgccatt gcactccagc ctggaggaca agagtgaaac
36661	tccattcccc tctgcaaaga aaaggaatat tatcagattc ctaagctttt tggctccccc
36721	tttagtttgg gggctggggt ggtgagtgtc tgacctggcc tcactgtcct ccctggatgt
36781	gatgagaccc aggtgtgggt caggatgtca ttcgtttgtc caccagaggg cgcccaaact
36841	gctttgagct gctgggaaat ggtgctccta gacttttagc aaacaaacaa aaaaaaatgg
36901	cacatcggca aatttcagac cattcttttt tttttttttt ttggttccag agtagctgaa
36961	atctttgttc agttacaagc aggataaaat ggaaactgcc tgggagaggc tgagaaacct
37021	tcttgcttgg gggaggtggg gcactgctag aattaatcgc ttcacagacc agcccatcca
37081	ggactcctca aatttggcaa aaaagccatt cattcattca ttcatttatg tagagacgag
37141	ggggatctgg ctatattgcc tagattggtc tcaaattcct ggcctcaagt gatcctcctg
37201	ccttggtcta ctaatgtgct gcgattacag gcatgagcca ccgtgcctag ctctagtgga
37261	cttgaaatgt tgccttgccc agggccctta tgttgaatgg cccaggtcca cttgtatggt
37321	tctgtaccaa ggttaacccc atcccataat gcctgggaca gttgatgcag gacaatcagc
37381	ttctgtgcca ttcaacctca ggactgagca tgctgggcat tgtggggtcc gaaggtggct
37441	cccctgtccc cttcaaaata ccctcttttt cttttcttct tttttttttt tttttttttt
37501	tgagacgaag tcttgctctg ttgccccagc tagagtgcag tggtgcgatc tcagctcccc
37561	gcaacctctg cttcccgggt tcaggcgatt ctcctgcctc agcctcctga gtagctggga
37621	ttacaggtgc ccaccgccac agctggctaa tttttgtatt tttagtagag acagggtttc
37681	accgtgttgg ccaggctggt cttgaactcc tgacctcagg caacctgccc acctcagcct
37741	cccaaagtgc tgggattaca ggtttgagcc actgggcctg gccttttttt tttttttttg
37801	agagggagtc tcactctgtt gcccaggctg gagtgcaatg gcgcgatctt gactcactgc
37861	aactccattt cccgggttca agtgattctc ctccctcagc ctcccaagta gctgggatta
37921	caggtgcatg ccaccacggc cagctaattt tgtattttta gtagagacag ggtttcacta
37981	tgttgatcat gctggtctca aactcctgac cttaggtgat ctgcccgcct tagcctccca
38041	aagtgttggg attacaggtg tgagccaccg cgcccagacc aaaatatgct cattttaata
38101	aaatgcacaa gtaggttgac aagaatttca cctgcaacct tgtcaaccac ctagaataaa
38161	agcctctgca gccctcccct aaagactcat caatgtgagg ctcaagaacc ttcttaggct
38221	gggctcggtg gctcatttct gtaatccctg cactttggaa ggctgaggca ggaggatctc
38281	ttgaggccag gagttcaaga caagcctggg caacatagcc agacctctgt ttctatcccc
38341	cacaaaaaga accttcttaa accggaattg agtcctacaa cctcgataac tcacaaataa
38401	gcccgtgtgg cctctcacag acttgggaag ttctccaagt gtccagggag atgtgccagg
38461	cgctttcctg ccgtgaccac cgtcctctgc ctgctccatt tcttggtggc cttcctttag
38521	acctgggcct cactcttgct tctctcctgc agctctgggc gacgttgctg gcagaggaaa
38581	tgagaagaag cccagtagcg tgagggctct gtccattgtc ctccccatcg gtaagcgcgg
38641	gccggtcccc cagcgtcccc caggtcacag cctcccgcta tgtgacctcg tgcctggctg
38701	gttgggcctg ttcacttttt ctcctggaca gggaacagcc ccactggtgt cctttatcac
38761	ccccacggcc tctcctggct tggggctgac agtgacaaga tcagacagct aaggggtcag
38821	atggaggatg tggagctggg tcccgtgctg tggaatagcc tcaccgagat ttgagtgcct
38881	tctggggaac tggttccctt gcagggggct gtgtggagag gcgcgctctc cctgcctcac
38941	ccatgctcat cctaactcgg ttaccatcac atctcttttt tctttttttc ttaaatttta
39001	agaaaaaaga aatttaattt ttttgagaga cagagtcttg ctctgtcacc caggctggag
39061	tgcagtggca ccatcatgcc tcgctgcagc ctcaatgtct gggctcaagc gatcctccca
39121	cctcagcctc ctgagtagct ggtgcaagcc actatacccc acttcctatt tcttaaaaag
39181	tcacagccct gtgtgtggct aatcctggac agaaatctag aagaagtcag ctacttctgg
39241	ggcgtggctc acccagtggg cttcaggtta gatatttctt atacttatga ggctgggtgt
39301	ggtggcttat gcctgtaatc ccagcacttt gggaggctga agtgggtgga ttgcttgggc
39361	tcaggagttc gagaccaacc tgggcaacat ggcgaaaccc tgtttctaga aaaggtacaa
39421	aaattagctg ggcaggtggc acgtgcctgt ggtaccagct acttgagggc ctgaggcagg
39481	aggatcgctt gaacctggga ggtcgaggtt gcagtgaact gagatcatgt cactgcactc
39541	cagcctggtg acagagcaag accccgtctc aaaaaaaaaa aaagaaagaa aaaaattctt
39601	atgcatagat ttgcctcttt tctgtttgtt tgttttgaga tggagtctcg ctctgtcgcc
39661	caggctggag tacagtggct caacctcggc tcactgcaac ctctgcctcc cgggttcaag
39721	caattctcct gcctcagcct cctgagtagc tgggactaca ggcgcccgcc accatgccca
39781	gctaattttt gtatttttag tagagactga ctgggtttca tcatgttggc caggctggtc
39841	tcgaactctt gacctcatga tccgcccgcc tcagcctccc aaaatgctgg gattacaggc
39901	gtgagccacc aggcccaggc cgcaaggcga tctctaaaca aacataaaag accaggagtc
39961	aaggttatgg tacgatgccc gtgttttcac tccagccacg gagctgggtc tctggtctcg
40021	ggggcagctg tgtgacagag cgtgcctctc cctacagtgc tcctcgtctt cctttgcctg
40081	ggggtcttcc ttctatggaa gaactggcgg cttaagaaca tcaacagcat caactttgac
40141	aaccccgtct atcagaagac cacagaggat gaggtccaca tttgccacaa ccaggacggc
40201	tacagctacc cctcggtgag tgaccctctc tagaaagcca gagcccatgg cggccccctc
40261	ccagctggag gcatatgatc ctcaagggac caggccgagg cttccccagc cctccagatc
40321	gaggacagca ttaggtgaat gcttctgtgc gctcattcag aatgtcagcg gacaatggcc
40381	ttggtggtgt agaggaatgt tggataagca aatagagagc tccatcagat ggtgacaggg
40441	caaagaaagt caaaaggagt tcagaggccg ggcgcggtgg ctcatgcctg taatcccagg
40501	actttgggag gccgaggctg gcggatcacc tgaagtcagg agtttgagac cagcttggcc
40561	atcatgacaa aaccccgtct ctattaaaaa tacaaaaaat tagccaggcg tgggagtggg
40621	cgcctgtaat cccagctact cgggaggccg aggtagaaaa atcgcttgaa cctaggaggc
40681	agaggttgca gtgagccgag atcgcgccac tgcattccag cccgggaggc aagagcaaaa
40741	ctccatctca aaaaaaaaaa aaaaaggagt tcagaggccc ggcatggtgg ttcacacatg
40801	tgatcccaga acttggggag gttgaggcag gagaatcacc tgagctcaga gttcaagacc
40861	agcctgggca gcacagcaag accccatctc tgcaaaaaat aaaaatttag cccagtgtgg
40921	tgatgagcgc ctagttccag ctactaggga ggctaaggca ggaggattgc ttgaggctaa
40981	ggtaggagat tgagactgca gtgacttgtg attgcgtcac tgcgctccag cctgggtgac
41041	agagcaagcc cttgtctctt aaaaaaaaaa aaaaattcaa agaagggttt ccagagggcc
41101	aggagggagg aagggagagg aggtgtttta tttttttgct tttatttttt attttgagac
41161	agagtctctc tctgtcaccc aggttggagt gcagtgctgt gatcttggct cactgcaact
41221	tctgcctcct gggttcaagc aattcttatg cctcagcctc agcctcctga gtagctggga
41281	ttacaacact atgcccgggt aatttttgta tttttagtag agacgaggtt tcgccatgtt
41341	gcccagactg gtctcgaact cctgacctca agtgatccac ccgccttggc ctccccacgt
41401	gctgggattg caggcgtgag ccactgcgcc cgccttgatc tttacacaag gggtttaggg
41461	taggtagcct tctctgaacc aggagaacag cctgtgcgaa ggccctgagg ctggaccgtg
41521	cctgttgggt ttgaggccgt tgtagctgga gcaaacagag agaggggtaa aaaggcagga
41581	ggctaccagg caggttgtgc agagccttgt gggccactgg ggaggacttt ggcttttgcc
41641	ctgagagcgg tgggaagtga ctgaatccgg tactcaccgt ctccctctgg cggctcctgg
41701	gggaacatgc ttggggatca ggctggggga ggctgccagg cccaggaggt gagaagtagg
41761	tggcctccag ccgtgtttcc tgaatgctgg actgatagtt tccgctgttt accatttgtt
41821	ggcagagaca gatggtcagt ctggaggatg acgtggcgtg aacatctgcc tggagtcccg
41881	tccctgccca gaacccttcc tgagacctcg ccggccttgt tttattcaaa gacagagaag
41941	accaaagcat tgcctgccag agctttgttt tatatattta ttcatctggg aggcagaaca
42001	ggcttcggac agtgcccatg caatggcttg ggttgggatt ttggtttctt cctttcctcg
42061	tgaaggataa gagaaacagg cccgggggga ccaggatgac acctccattt ctctccagga
42121	agttttgagt ttctctccac cgtgacacaa tcctcaaaca tggaagatga aaggggaggg
42181	gatgtcaggc ccagagaagc aagtggcttt caacacacaa cagcagatgg caccaacggg
42241	accccctggc cctgcctcat ccaccaatct ctaagccaaa cccctaaact caggagtcaa
42301	cgtgtttacc tcttctatgc aagccttgct agacagccag gttagccttt gccctgtcac
42361	ccccgaatca tgacccaccc agtgtctttc gaggtgggtt tgtaccttcc ttaagccagg
42421	aaagggattc atggcgtcgg aaatgatctg gctgaatccg tggtggcacc gagaccaaac
42481	tcattcacca aatgatgcca cttcccagag gcagagcctg agtcactggt cacccttaat
42541	atttattaag tgcctgagac acccggttac cttggccgtg aggacacgtg gcctgcaccc
42601	aggtgtggct gtcaggacac cagcctggtg cccatcctcc cgacccctac ccacttccat
42661	tcccgtggtc tccttgcact ttctcagttc agagttgtac actgtgtaca tttggcattt
42721	gtgttattat tttgcactgt tttctgtcgt gtgtgttggg atgggatccc aggccaggga
42781	aagcccgtgt caatgaatgc cggggacaga gaggggcagg ttgaccggga cttcaaagcc
42841	gtgatcgtga atatcgagaa ctgccattgt cgtctttatg tccgcccacc tagtgcttcc
42901	acttctatgc aaatgcctcc aagccattca cttccccaat cttgtcgttg atgggtatgt
42961	gtttaaaaca tgcacggtga ggccgggcgc agtggctcac gcctgtaatc ccagcacttt
43021	gggaggccga ggcgggtgga tcatgaggtc aggagatcga gaccatcctg gctaacacgt
43081	gaaaccccgt ctctactaaa aatacaaaaa attagccggg cgtggtggcg ggcacctgta
43141	gtcccagcta ctcgggaggc tgaggcagga gaatggtgtg aacccgggaa gcggagcttg
43201	cagtgagccg agattgcgcc actgcagtcc gcagtctggc ctgggcgaca gagcgagact
43261	ccgtctcaaa aaaaaaaaac aaaaaaaaac catgcatggt gcatcagcag cccatggcct
43321	ctggccaggc atggcgaggc tgaggtggga ggatggtttg agctcaggca tttgaggctg
43381	tcgtgagcta tgattatgcc actgctttcc agcctgggca acatagtaag accccatctc
43441	ttaaaaaatg aatttggcca gacacaggtg cctcacgcct gtaatcccag cactttggga
43501	ggctgagctg gatcacttga gttcaggagt tggagaccag gcctgagcaa caaagcgaga
43561	tcccatctct acaaaaacca aaaagttaaa aatcagctgg gtacggtggc acgtgcctgt
43621	gatcccagct acttgggagg ctgaggcagg aggatcgcct gagcccagga ggtggaggtt
43681	gcagtgagcc atgatcgagc cactgcactc cagcctgggc aacagatgaa gaccctattt
43741	cagaaataca actataaaaa aataaataaa tcctccagtc tggatcgttt gacgggactt
43801	caggttcttt ctgaaatcgc cgtgttactg ttgcactgat gtccggagag acagtgacag
43861	cctccgtcag actcccgcgt gaagatgtca caagggattg gcaattgtcc ccagggacaa
43921	aacactgtgt cccccccagt gcagggaacc gtgataagcc tttctggttt cggagcacgt
43981	aaatgcgtcc ctgtacagat agtggggatt ttttgttatg tttgcacttt gtatattggt
44041	tgaaactgtt atcacttata tatatatata tacacacata tatataaaat ctatttattt
44101	ttgcaaaccc tggttgctgt atttgttcag tgactattct cggggccctg tgtagggggt
44161	tattgcctct gaaatgcctc ttctttatgt acaaagatta tttgcacgaa ctggactgtg
44221	tgcaacgctt tttgggagaa tgatgtcccc gttgtatgta tgagtggctt ctgggagatg
44281	ggtgtcactt tttaaaccac tgtatagaag gtttttgtag cctgaatgtc ttactgtgat
44341	caattaaatt tcttaaatg

SEQ ID NO: 31: Human LDL receptor cDNA sequence (NM_—000527)
Coding Region Underlined.