Method for Production of Transfected Cells

The present invention relates to a method for production of transfected cells, more particularly, a method which makes possible to transfer a foreign gene into target cells with high efficiency in the fields such as cell technology, genetic engineering, developmental engineering and the like.

As a method for transferring a foreign gene into target cells, there are known a calcium phosphate method, a DEAE-dextran method, a liposome method, an electroporation method, a microinjection method, a particle gun method and the like. All of these methods have both advantages and disadvantages with respect to manipulation procedures, efficiency, damage on cells and the like. Among these methods, a perforation method such as an electroporation method, a microinjection method, a particle gun method and the like enables easy handling of the cells without using special reagents and provides good transfer efficiency. However, damage to cells by perforation cannot be avoided.

The object of the present invention is to provide a method for improving the transfer efficiency when a foreign gene is transferred into target cells by a perforation method to produce transfected cells.

The first aspect of the present invention relates to a method for the production of transfected cells, which is characterized by comprising a step of culturing the cells in the presence of a cell-adhesive substance, after injection of a foreign gene into target cells using a perforation method.

The second aspect of the present invention relates to transfected cells containing a foreign gene which are produced by the method of the present invention.

The third aspect of the present invention relates to a kit, which is used for a method for the production of transfected cells according to the first aspect of the present invention and is characterized by containing a cell-adhesive substance.

FIG. 1 is a graph showing the effect of cell-adhesive polypeptide treatment on gene transfer efficiency in the transfer of pCAT-control vector into human epidermoid cancer cell A-431.

FIG. 2 is a graph showing the effect of cell-adhesive polypeptide treatment on gene transfer efficiency in the transfer of pCAT-control vector into African green monkey kidney cell COS-7-7.

The method of the present invention is characterized by culturing the cell in the presence of a substance having the cell-adhesive activity after a foreign gene is transferred into target cells using a perforation method.

As used herein, the perforation method means a method for injection of a gene by perforating a cell wall, including an electroporation method, a microinjection method, a particle gun method and the like. The electroporation method is as described in, for example, Tanpakushitsu, Kakusan, Koso, volume 31, pages 1591-1603 (1986). The microinjection method is as described in, for example, Cell, volume 22, pages 479-488 (1980). The particle gun method is as described in, for example, Technique, volume 3, pages 3-16 (1991). These methods include the known methods used for transferring a gene into cells.

As cells to be used in these perforation methods, for example, there are animal cells which are prepared according to a known method ›"Shin-Seikagaku Jikkenkoza 18, Saibobaiyogijyutsu", 1st edition (1990), edited by Nippon Seikagakugakkai, published by Tokyo Kagakudojin! and cultured animal cells may be used.

As used herein, a cell-adhesive substance refers to a substance having the cell-adhesive, that is, the activity to make target cells adhere to a cell, to an extracellular matrix which is a substance filling a space between cells in the tissue, or to a material such as plastic, glass and the like. In the present invention, any substances having the activity can be used as long as they give no adverse effects on transfection of target cells. Such the activity is to fix cells, for example, to culture ware covered with a cell-adhesive substance while maintaining the cell in its form, or in a spreaded form, that is, in the changed form after the cell has been spreaded in one or more directions.

Attachment between the cell-adhesive substance and the target cell can be assayed using a conventional method. The method includes, for example, a method described in Nature, 352:438-441 (1991). Briefly, the cell-adhesive substance covers a plastic dish, and a population of cells to be assayed is put into a medium and allowing it to stand for 30 minutes to 2 hours. After this incubation period, non-adhered cells are recovered, counted and assayed for viability. Cells adhered to the cell-adhesive substance are recovered using trypsin or a cell dissociation buffer (for example, Gibco), counted and assayed for viability. Then, a ratio of adhered cells is calculated and compared with a standard control such as a plastic dish covered with bovine serum albumin (BSA). A combination of cell-adhesive substance/cell can be determined by substantial adhesion of the target cell with the cell-adhesive substance assayed. In addition, the cell-spreading activity can be determined by observing a change in the form of the adhered cells under a microscope before dissociating cells using trypsin or a cell dissociation buffer, in the above procedures.

Examples of the cell-adhesive substance include, for example, a cell-adhesive polypeptide or a functional equivalent thereof and a cell-adhesive synthetic polymer.

Examples of the polypeptide to be used in the present invention, having the cell-adhering activity include a cell-adhesive polypeptide such as invasin, polylysine which is not derived from an extracellular matrix, a polypeptide showing the cell-spreading activity described in JP-A 2-311498, components of an extracellular matrix such as fibronectin, laminin, collagen, vitronectin, osteopontin, thrombospondin, tenascin and the like. The extracellular matrix components can be prepared from a natural or cultured source by a known method ›International Journal of Cancer, volume 20, pages 1-5 (1977); Journal of Biological Chemistry, volume 254, pages 9933-9937, (1979); "Zoku-Seikagaku Jikkenkoza", volume 6, Saibokokkaku no Kozo to Kino (Structure and Function of Cell Skeleton) (last volume), (1st edition) (1986) edited by Nippon Seikagakugakkai, published by Tokyo Kagakudojin; Cell Structure and Function, volume 13, pages 281-292 (1988); Journal of Biological Chemistry, volume 264, pages 18202-18208 (1989); and Journal of Biological Chemistry, volume 260, pages 12240-12245 (1985)!. The cell-adhesive polypeptide may be substantially purified extracellular matrices exhibiting the cell-adhering activity, substantially purified extracellular matrix fragments or a mixture thereof. More particularly, proteins and polypeptides having the cell-adhesive or the cell spreading activity, or a functional equivalent thereof, may be used.

As these cell-adhesive polypeptides, substantially purified natural polypeptides, polypeptides from enzymatical or chemical degradation of the natural polypeptides, or the similar polypeptides made by genetic engineering may be used. Further, materials obtained by modifying these polypeptides without impairing the function, that is, the cell-adhering activity or the cell-spreading activity may be used. In the present invention, even if the polypeptide has a deletion, substitution, addition and/or insertion of amino acids in the amino acid sequence of a polypeptide from natural origin, as long as the polypeptide has the desired cell-adhering activity or the cell-spreading activity, it is referred to as a functional equivalent of a polypeptide having the natural amino acid sequence. That is, it is known that naturally occurring proteins include proteins of which amino acid sequences have mutations such as deletions, insertions, additions, substitutions and the like of amino acids, which are due to a modification reaction in the living body after production or during purification, in addition to polymorphism or mutation of genes encoding those naturally occurring proteins. Regardless of these mutations, there are proteins exhibiting the physiological and biological activity substantially equivalent to that of proteins having no mutation. Like this, even when there is a structural difference between polypeptides, as long as they share the common main functions, they are called polypeptides having the functionally equivalent activity.

This is also true where the above mutations are artificially introduced into the amino acid sequence of proteins. In this case, a greater variety of mutants may be made. As long as these mutants exhibit the physiological activity substantially equivalent to that of proteins having no mutations, they are interpreted to be a polypeptide having the functionally equivalent activity.

For example, in many cases, a methionine residue which is present at a N-terminus of a protein expressed in Escherichia coli is said to be removed by action of methionine aminopeptidase, thus, generating both proteins having a methionine residue or those having no methionine residue depending upon the kind of proteins. However, whether or not a protein has a methionine residue does not affect the protein activity in many cases. In addition, it is known that a polypeptide, a certain cysteine residue of which is substituted with a serine residue in the amino acid sequence of human interleukin-2 (IL-2), retains the interleukin-2 activity ›Science, volume 224, page 1431 (1984)!.

Further, upon production of proteins by genetic engineering, proteins are frequently expressed as fused proteins. For example, in order to increase an amount of an expressed protein of interest, the protein is expressed by adding a N-terminal peptide chain derived from another protein to the N-terminal of the protein of interest, or adding a suitable peptide chain to the N-terminus or the C-terminus of the protein of interest to facilitate purification of the protein of interest by using a carrier having the affinity to the added peptide chain.

In this respect, the related biotechnological 10 techniques have advanced to a state in which deletion, substitution, addition or other modification of amino acids in a functional region of the polypeptide can be routinely carried out. Then, the resulting amino acid sequence may be routinely screened for the desired cell-adhering activity or the cell-spreading activity according to the above method.

Polypeptides having the cell-adhering activity may be an artificial polypeptide containing, in the molecule, the amino acid sequence necessary for the cell-adhering activity, for example, the amino acid sequence may be selected from the amino acid sequence represented by SEQ ID No:1 (RGDS), the amino acid sequence represented by SEQ ID NO:2 (CS1) and the amino acid sequence represented by SEQ ID NO:6 (central sequence of laminin, YIGSR). These polypeptides can be prepared in a large amount by a genetic engineering method or a chemical synthesis method and may be used as a purified polypeptide.

Examples of the artificial polypeptide having, in the molecule, the amino acid sequence represented by SEQ ID NO:1 include a polypeptide represented by SEQ ID NO:7 described in JP-A 1-180900. The polypeptide can be prepared using Escherichia coli HB101/pTF1409 (FERM BP-1939) according to a method described in JP-A 1-180900. In addition polypeptides represented by their respective sequence ID numbers in the sequence list shown in Table 1 below can be prepared according to a genetic engineering method described in each specification.

In addition, a plasmid pCHV90 contained in Escherichia coli HB101/pCHV90 in Table 1 can be prepared using Escherichia coli HB101/pHD101 (FERM BP-2264) and Escherichia coli JM109/pTF7021 (FERM BP-1941) according to a method described in JP-A 5-271291.

Alternatively, artificial polypeptides having, in the molecule, the amino acid sequence represented by SEQ ID NO:1 can be PolyRGDS described, described in JP-A 3-173828, can be synthesized and used.

Examples of artificial polypeptides having, in the molecule, the amino acid sequence represented by SEQ ID NO:2 include a polypeptide represented by SEQ ID NO:4, described in JP-A 2-311498, and the polypeptide can be prepared by genetic engineering using Escherichia coli HB101/pHD102 (FERM P-10721) according to a method described in JP-A 2-311498. In addition, a polypeptide represented by SEQ ID NO:2 may be chemically synthesized according to a method described in JP-A 3-284700.

Further, examples of artificial polypeptides having, in the molecule, the amino acid sequence represented by SEQ ID NO:2 and the amino acid sequence represented by SEQ ID NO:3 include a polypeptide represented by SEQ ID NO:21 described in JP-A 2-311498 and the polypeptide can be prepared by genetic engineering using Escherichia coli HB101/pCH102 (FERM BP-2800) according to a method described in JP-A 2-311498. In addition, a polypeptide represented by SEQ ID NO:5 described in JP-A 3-284700 is a polypeptide containing, in the molecule, the amino acid sequences of SEQ ID NOs: 1 and 2 and the polypeptide can be prepared by genetic engineering using Escherichia coli HB101/pCS25 (FERM P-11339) according to a method described in JP-A 3-284700.

As described above, examples of the polypeptides used in the present invention are cell-adhesive polypeptides containing, in the molecule, the amino acid sequence represented by SEQ ID NO:1 and/or the amino acid sequence represented by SEQ ID NO:2. As the polypeptide, a polypeptide obtained by covalently binding a polypeptide derived from a cell adhesion domain of human fibronectin ›"Fibronectin", pages 47-121 (1989), edited by Mosher, D. F., published by Academic Press! with a CS1 polypeptide derived from the same (ibid), a polypeptide derived from a heparin binding domain (ibid) containing a CS1 polypeptide, or a polypeptide derived from a cell adhesion domain can be used, and they can be made by genetic engineering, respectively. For example, respective necessary regions are taken out from a vector containing a DNA encoding a cell adhesion domain-derived polypeptide, a vector containing a DNA encoding a CS1 polypeptide, and a vector containing a DNA encoding a heparin binding domain-derived peptide containing a CS1 polypeptide, respectively, and they can be used alone or in combination thereof to make a vector expressing a polypeptide containing, in the molecule, the amino acid sequence represented by SEQ ID NO:1 and/or the amino acid sequence represented by SEQ ID NO:2.

When a polypeptide, where a polypeptide containing, in the molecule, the amino acid sequence represented by SEQ ID NO:1 and a polypeptide containing, in the molecule, the amino acid sequence represented by SEQ ID NO:2 are covalently bound, is made, a covalent bonding between polypeptides may be a direct bonding or an indirect bonding, for example, an indirect bonding via a spacer. A spacer is an insertion sequence for adjusting an intermolecular distance in each region. As the spacer, an arbitrary peptide chain can be used, for example, a sequence upstream of a CS1 region in the fibronectin molecule. The spacer sequence can be easily introduced therein by genetic engineering.

The cell-adhesive synthetic polymers include the known poly-N-p-vinylbenzyl-D-lactoneamide (PVLA) polymer.

In the present invention, the target cell include, but is not limited to, hematopoietic stem cell, peripheral blood stem cell, umbilical blood cell, ES cell, lymphocyte, cancer cell and the like.

Examples of the foreign gene include, but are not limited to, nucleic acid selected from nucleic acids encoding proteins, nucleic acids encoding polypeptides, antisense DNAs, antisense RNAs, ribozymes, nucleic acids encoding intracellular antibodies and pseudogenes (decoy genes). In the present invention, the foreign gene may be inserted into a vector.

Examples of the vector are retrovirus vector, adenovirus vector, vacciniavirus vector, herpesvirus vector and the like.

According to the present invention a transfected cell with a foreign gene can be obtained with high efficiency by culturing a target cell, into which a foreign gene has been transferred by a perforation method according to a conventional method, in the presence of a cell-adhesive substance. A cell culture method may be selected from the known methods depending upon the cell used. For example, when cell culturing is performed in the presence of a cell-adhesive polypeptide, 250 to 2000 .mu.g/ml of the cell-adhesive polypeptide may be used in a culture medium to culture it according to a conventional method.

Particularly, culturing is preferably carried out using a culture ware covered with a cell-adhesive substance. The culture ware refers to any ware normally used for cell culture, for example, a culture dish, a culture ware using a microcarrier, and a culture ware using fibrous hollow fibers. The culture ware may be covered with the substance by coating or spraying. For example, the culture may be easily covered with the polypeptide by dissolving it in a suitable solution such as a phosphate buffered saline (PBS), adding the solution to the culture ware and allowing it to stand for a suitable period of time. An amount of the polypeptide with which the culture ware is covered may be selected from a range of 50 to 1000 pmol/cm.sup.2, suitably 150 to 600 pmol/cm.sup.2.

Transfected cells which have been cultured in the presence of the cell-adhesive substance can be obtained from a culture according to a conventional method. Thus, transfected cells can be produced with high efficiency.

The resulting transfected cells are useful for the production of useful substances by cells using recombinant DNA techniques, development of disease models, gene therapy and the like. Thus, transfected cells can be produced with high efficiency according to the present invention.

In addition, the present invention can be simply carried out by using a kit containing a cell-adhesive substance. The cell-adhesive substance to be contained in the kit may be in a form of solutions or lyophilized powders. The kit may contain a buffer for dissolving or diluting the cell-adhesive substance, a cell culture medium, a cell culture ware and the like. For example, a transfected cell can be simply produced by preparing a kit combining polypeptides, PBS for diluting the polypeptide, a cell culture ware and the like which are used for the method of the present invention. A reagent contained in the kit may be in liquid form or in lyophilized form.

A perforation method in the present invention can be used by appropriately selecting from an electroporation method, a microinjection method, a particle gun method and the like depending upon the purpose.

The present invention is illustrated by the non-limiting Examples below.

1. Coating of culture dish with cell-adhesive polypeptide.

A polypeptide represented by SEQ ID NO:3 (hereinafter referred to as "C274"), a polypeptide represented by SEQ ID NO:4 (hereinafter referred to as H296) and a polypeptide represented by SEQ ID NO:5 (hereinafter referred to as "C.multidot.CS1") were each dissolved in a phosphate buffered saline (PBS) to 1 .mu.M, respectively, which were then sterilized using a 0.22 .mu.m filter (Millex-GV, Millipore).

Each 1 ml/well of these solutions was added to a 24-well polystyrene culture dish (manufactured by Corning), respectively, to coat the dish at 4.degree. C. overnight. These dishes were rinsed with a 500.mu.l/well of a Dulbecco's modified minimum basal medium not containing bovine fetal serum prior to the addition of a transformed cell described below.

2. Transfection of cells

Two culture dishes (diameter: 100 mm) of human epidermoid cancer cell A-431 which had been cultured in a Dulbecco's modified minimum basal medium containing 10% bovine fetal serum were rinsed with 10 ml of a Dulbecco's modified minimum basal medium not containing bovine fetal serum, respectively, and 3 ml of PBS containing 0.25% bovine trypsin and 0.02% EDTA was added thereto to detach cells from the culture dish. To these, 7 ml of a Dulbecco's modified minimum basal medium not containing bovine fetal serum was added, followed by centrifugation at 800 rpm for 3 minutes to collect cells. The resulting cells were suspended in 10 ml of a Dulbecco's modified minimum basal medium containing bovine fetal serum, followed by centrifugation at 800 rpm for 3 minutes to collect cells. The resulting cells were combined, suspended in 10 ml of PBS, a 3/10 aliquot of the suspension was taken and divided into two equal aliquots, which were centrifuged at 800 rpm for 3 minutes to collect cells, respectively. The resulting cells were suspended again in 10 ml of PBS, followed by centrifugation at 800 rpm for 3 minutes to collect two batches of cells. One batch of the resulting cells were suspended in 1 ml of PBS containing 15 .mu.g of pCAT-control vector (Promega) which had been aseptically prepared, and placed in an electroporation cuvette for Gene Pulser (BioRad), which was allowed to stand in ice for 10 minutes. The other batch of the resulting cells were suspended in 1 ml of PBS, and placed in an electroporation cuvette for Gene Pulser (BioRad), which was allowed to 15 stand in ice for 10 minutes. Each batch of cells was allowed to stand in ice for 10 minutes, and voltage was applied thereto at 250 V and 960 .mu.F. After application, the cells were allowed to stand in a cuvette in ice for 10 minutes. Thereafter, the cells were recovered into 15 ml 20 of a Dulbecco's modified minimum basal medium containing 10% bovine fetal serum, 1 ml/well of which were added to a 24-well polystyrene culture dish covered with the above polypeptide. These cells were cultured at 37.degree. C. in the presence of 5% CO.sub.2 gas overnight, the medium was removed by aspiration, and 1 ml/well of a fresh Dulbecco's modified minimum basal medium containing 10% bovine fetal serum was added thereto, followed by culturing at 38.degree. C. in the presence of 5% CO.sub.2 gas overnight.

3. Determination of transfection efficiency (efficiency of gene transfer)

The cultured cells were rinsed three times with 1.25 ml of PBS per well, a lysed cell solution was prepared, and detection of expressed CAT was carried out using CAT-ELISA kit (manufactured by Boehringer Mannheim) according to a method for using the present kit. Since the present kit used a horseradish peroxidase-labelled secondary antibody and ABTS as a substrate, a ratio of absorbance at 405 nm/490 nm was determined. A value obtained by subtracting a blank value from a value for each group in a case of addition of pCAT-control vector, using a value for the group in a case of no addition of pCAT-control vector upon electroporation as a blank, was adopted as an amount of expressed CAT.

The results thereof are shown in FIG. 1. That is, FIG. 1 is a figure showing efficiency of gene transfer into a cell in each polypeptide-treatment group, where the ordinate shows non-treated group and each polypeptide-treatment group and the abscissa shows gene transfer efficiency expressed as a ratio of absorbance at 405 nm relative to that at 490 nm.

As shown in FIG. 1, an amount of expressed CAT in the culture dish in the C274, H296 or C.multidot.CS1-treatment group is higher as compared with that in a non-treatment group, demonstrating that efficiency of transfer of pCAT-control vector into a cell is higher.

1. Coating of culture dish with cell-adhesive polypeptide

A polypeptide represented by SEQ ID NO:3 (hereinafter referred to as "C274"), a polypeptide represented by SEQ ID NO:4 (hereinafter referred to as "H296") and a polypeptide represented by SEQ ID NO:5 (hereinafter referred to as "C.multidot.CS1") were each dissolved in a phosphate buffered saline (PBS) to 1 .mu.M, respectively, which were then sterilized using a 0.22 .mu.m filter (Millex-GV, Millipore). 1 ml/well of these solutions were added to a 24-well polystyrene culture dish (manufactured by Corning) to coat the dish at 4.degree. C. overnight, respectively. These dishes were rinsed with 500 .mu.l/well of a Dulbecco's modified minimum basal medium not containing bovine fetal serum prior to addition of a transformed cell described below.

2. Transfection of cell

Two culture dishes (diameter: 100 mm) of African green monkey kidney cell COS-7 which had been cultured in a Dulbecco's modified minimum basal medium containing 10% bovine fetal serum were rinsed with 10 ml of a Dulbecco's modified minimum basal medium not containing bovine fetal serum, respectively, and 3 ml of PBS containing 0.25% bovine trypsin and 0.02% EDTA was added thereto to detach cells from the culture dish. To these, 7 ml of a Dulbecco's modified minimum basal medium not containing bovine fetal serum was added, respectively, followed by centrifugation at 800 rpm for 3 minutes to collect cells. The resulting cells were suspended in 10 ml of a Dulbecco's modified minimum basal medium containing bovine fetal serum, followed by centrifugation at 800 rpm for 3 minutes to collect cells. The resulting cells were combined, suspended in 12 ml of PBS, a 5/6 aliquot of the suspension was taken and divided into two equal aliquots, which were centrifuged at 800 rpm for 3 minutes to collect cells, respectively. The resulting cells were suspended in 6 ml of PBS, followed by centrifugation at 800 rpm for 3 minutes to collect two batches of cells. One batch of the resulting cells were suspended in 1 ml of PBS containing 15 .mu.g of pCAT-control vector (Promega) which had been aseptically prepared, and placed in an electroporation cuvette for Gene Pulser (BioRad), which was allowed to stand in ice for 10 minutes. The other batch of the resulting cells were suspended in 1 ml of PBS, and placed in an electroporation cuvette for Gene Pulser (BioRad), which was allowed to stand in ice for 10 minutes. Each batch of cells was allowed to stand in ice for 10 minutes, and voltage was applied thereto at 250 V and 960 .mu.F. After application, the cells were allowed to stand in a cuvette in ice for 10 minutes. Thereafter, the cells were recovered into 15 ml of a Dulbeccol's modified minimum basal medium containing 10% bovine fetal serum, 1 ml/well of the cells were added to a 24-well polystyrene culture dish covered with the above polypeptide. These cells were cultured at 37.degree. C. in the presence of 5% CO.sub.2 gas overnight, the medium was removed by aspiration, and 1 ml/well of a fresh Dulbecco's modified minimum basal medium containing 10% bovine fetal serum was added, followed by culturing at 37.degree. C. in the presence of 5% CO.sub.2 gas overnight.

3. Determination of transfection efficiency (efficiency of gene transfer)

The cultured cells were rinsed three times with 1.25 ml of PBS per well, a lysed cell solution was prepared, and detection of expressed CAT was carried out using CAT-ELISA kit (manufactured by Boehringer Mannheim) according to a method for using the present kit. Since the present kit used a horseradish peroxidase-labelled secondary antibody and ABTS as a substrate, a ratio of absorbance at 405 nm/490 nm was determined. A value obtained by subtracting a blank value from a value for each group in a case of addition of pCAT-control vector, using a value for the group in a case of no addition of pCAT-control vector upon electroporation as a blank, was adopted as an amount of expressed CAT. The results thereof are shown in FIG. 2. That is, FIG. 2 is a figure showing efficiency of gene transfer into a cell in each polypeptide-treatment group, where the ordinate shows non-treated group and each polypeptide-treatment group and the abscissa shows gene transfer efficiency expressed as a ratio of absorbance at 405 nm relative to that at 490 nm.

As shown in FIG. 2, an amount of expressed CAT in the culture dish in the above C274, H296 or C.multidot.CS1-treatment group is higher as compared with that in a non-treatment group, demonstrating that efficiency of transfer of pCAT-control vector into a cell is higher.

Preparation of kit

A kit for production of gene-transferred cells was made from C274, H296, C.multidot.CS1, PBS and a culturing dish as shown in Table 2 below. Reagents A, B and C were prepared so that the above polypeptides were adjusted with PBS to the indicated concentrations shown in the Table. Other components used are described in Example 1. In addition, all reagents A, B and C and a diluent for the reagents were aseptically prepared by pre-filtering with a 0.22 .mu.m sterile filter.

As described above, the present invention can overcome the problems of the previous methods for gene transfer into cells and provide a method, for production of transfected cells, having improved efficiency of gene transfer into target cells. The present invention can also provide a kit, for production of transfected cells, which is used for the method.