US 6,083,690 AGrant

Methods and Compositions for Identifying Osteogenic Agents

Issue Date:2000-07-04

•18 Claims

•26 Drawing Sheets

Abstract

Methods and compositions for identifying osteogenic agents are disclosed, wherein a bone morphogenetic protein promoter is utilized in an assay system to modulate the production of an assayable product of a reporter gene.

Metadata

Assignee

Osteoscreen, Inc.

Inventors

Stephen E. Harris
Gregory R. Mundy
Nandini Ghosh-Choudhury
Jian Q. Feng

Application Information

Application Number:US 4584341

Filing Date:1995-06-02

Priority Date:1995-06-02

Art Unit:162

Classifications

IPC:

C12Q 168C12Q 102C07H 2104C12N 1585

Field of Search:

4355364;6;29;172.3;320.1;325;375;440;45523.1;24.1

Patent Drawings (26 sheets)

Description

TECHNICAL FIELD

The present invention relates to assay techniques for identifying agents which modulate bone growth.

BACKGROUND OF THE INVENTION

Although there is a great deal of information available on the factors which influence the breakdown and resorption of bone, information on growth factors which stimulate the formation of new bone is more limited. Investigators have searched for sources of such activities and have found that bone tissue itself is a storehouse for factors which have the capacity for stimulating bone cells. Thus, extracts of bovine tissue obtained from slaughterhouses contain not only structural proteins which are responsible for maintaining the structural integrity of bone, but also biologically active bone growth factors which can stimulate bone cells to proliferate. Among these latter factors are transforming growth factor .beta., the heparin-binding growth factors (acidic and basic fibroblast growth factor), the insulin-like growth factors (insulin-like growth factor I and insulin-like growth factor II) and a recently described family of proteins called bone morphogenetic proteins (BMPs). All of these growth factors have effects on other types of cells as well as on bone cells.

The BMPs are novel factors in the extended transforming growth factor .beta. family. They were first identified in extracts of demineralized bone (Urist 1965, Wozney et al., 1988). Recombinant BMP-2 and BMP-4 can induce new bone formation when they are injected locally into the subcutaneous tissues of rats (Wozney 1992, Wozney & Rosen 1993). These factors are expressed by normal osteoblasts as they differentiate, and have been shown to stimulate osteoblast differentiation and bone nodule formation in vitro as well as bone formation in vivo (Harris et al., 1994). This latter property suggests potential usefulness as therapeutic agents in diseases which result in bone loss.

The cells which are responsible for forming bone are osteoblasts. As osteoblasts differentiate from precursors to mature bone-forming cells, they express and secrete a number of the structural proteins of the bone matrix including Type-1 collagen, osteocalcin, osteopontin and alkaline phosphatase (Stein et al, 1990, Harris et al, 1994). They also synthesize a number of growth regulatory peptides which are stored in the bone matrix and are presumably responsible for normal bone formation. These growth regulatory peptides include the BMPs (Harris et al, 1994). In studies of primary cultures of fetal rat calvarial osteoblasts, BMPs 1, 2, 3, 4, and 6 are expressed by cultured cells prior to the formation of mineralized bone nodules (Harris et al, 1994). Expression of the BMPs coincides with expression of alkaline phosphatase, osteocalcin and osteopontin.

Although the BMPs have powerful effects to stimulate bone formation in vitro and in vivo, there are disadvantages to their use as therapeutic agents to enhance bone healing. Receptors for the bone morphogenetic proteins have been identified in many tissues, and the BMPs themselves are expressed in a large variety of tissues in specific temporal and spatial patterns. This suggests that they may have effects on many tissues other than bone, potentially limiting their usefulness as therapeutic agents when administered systemically. Moreover, since they are peptides, they would have to be administered by injection. These disadvantages are severe limitations to the development of BMPs as therapeutic agents.

It is an object of the present invention to overcome the limitations inherent in known osteogenic agents by providing a method to identify potential drugs which would stimulate production of BMPs locally in bone.

PRIOR ART

Sequence data on small fragments of the 5'-flanking region of the BMP-4 gene have been published (Chen et al, 1993; Kurihara et al, 1993), but the promoter has not been previously functionally identified or isolated.

DISCLOSURE OF THE INVENTION

A cell-based assay technique for identifying and evaluating compounds which stimulate the growth of bone is provided, comprising culturing a host cell line comprising an expression vector comprising a DNA sequence encoding a promoter region of at least one bone morphogenetic protein, operatively linked to a reporter gene encoding an assayable product under conditions which permit expression of said assayable product, contacting the cultured cell line with at least one compound suspected of possessing osteogenic activity, and identifying osteogenic agents by their ability to modulate the expression of the reporter gene and thereby increase the production of the assayable product.

This assay technique specifically identifies osteogenic agents which stimulate bone cells to produce bone growth factors in the bone morphogenetic protein family. These osteogenic agents display the capacity to increase the activity of the promoters of genes of members of the BMP family and other bone growth factors normally produced by e.g. bone cells.

Also provided in accordance with the present invention are isolated DNA sequences encoding a promoter region of at least one bone morphogenetic protein, and a system for identifying osteogenic agents comprising an expression vector comprising such promoter sequences operatively linked to a reporter gene encoding an assayable product, and means for detecting the assayable product produced in response to exposure to an osteogenic compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A graphically depicts a restriction enzyme map of mouse genomic BMP-4 and a diagram of two transcripts. The mouse BMP-4 gene transcription unit is .about.7 kb and contains 2 coding exons (closed boxes) and 3 non-coding exons, labeled exons 1A, 1B and 2. This 19 kb clone has an .about.6 kb 5'-flanking region and an .about.7 kb 3'-flanking region. The diagram shows approximately 2.4 kb of the 5'-flanking region, and a small region of the 3'-flanking region. The lower panel shows two alternative transcripts of BMP-4. Both have the same exons 2, 3 and 4 but a different exon 1. Transcript A has exon 1A and transcript B has exon 1B whose size was estimated according to RT-PCR and primer extension analysis in FRC cells;

FIG. 1B depicts the DNA sequence of selected portions of mouse genomic BMP-4 (SEQ. ID NO. 1) and the predicted amino acid sequences of the identified coding exons (SEQ. ID NO. 2). The numbers on the right show the position of the nucleotide sequence and the bold numbers indicate the location of the amino acid sequence of the coding region. Most of the coding sequence is in exon 4. The end of the transcription unit was estimated based on a 1.8 kb transcript. Primer 1 in exon 1A was used in RT-PCR analysis with Primer 3 in exon 3. Primer 2 in exon 1B was used in RT-PCR analysis with Primer 3. Primer B1 and B2 were used in primer extension reactions;

FIG. 1C portrays the sequence of the BMP-4 exon 1A 5'-flanking region and potential response elements in the mouse BMP-4 1A promoter (SEQ. ID NO. 3). The sequences of 2688 bp of the mouse BMP-4 gene are shown. Nucleotides are numbered on the left with +1 corresponding to the major transcription start site of the 1A promoter. The response elements of DR-1A Proximal and DR-1A Distal oligonucleotides are indicated. The other potential response DNA elements in the boxes are p53, RB (retinoblastoma), SP-1, AP-1, and AP-2. Primer A, indicated by the line above the DNA sequence at +114 to +96, was used for primer extension analysis of exon 1A-containing transcripts;

FIG. 2 depicts the results of a primer extension assay. Total RNAs prepared from FRC cells (on the left frame) and mouse embryo 9.5 days (on the right) were used with primer A or the complement of primer 2 . Two major extended fragments, 67 and 115 bp, indicated in lane A were obtained from primer A. Two 1B primers, primer B1 and primer B2, also gave negative results with both FRC and mouse embryo total RNA as template. Transcript B is not detectable with this assay. By RT-PCR, transcript B can be detected and quantified;

FIG. 3A is a photographic representation of gel electrophoresis of 1A-3 and 1B-3 RT-PCR products of the BMP-4 gene. RT-PCR was performed with two pairs of primers using FRC cell poly A.sup.+ mRNA as the template. The products were verified by the DNA sequence;

FIG. 3B is a schematic diagram of spliced BMP-4 RT-PCR products with 1A and 1B exons in FRC cells. RT-PCR was performed with two pairs of primers using FRC cell poly A.sup.+ mRNA as the template. The diagram shows where the primers are located in the BMP-4 genomic DNA. RT-PCR product 1A-2-3 which contains exon 1A, exon 2 and the 5' region of exon 3, was produced with primer 1 and primer 3. Primer 2 and primer 3 generated two RT-PCR products with the exon 1B-2-3 pattern. The heterogeneity in size of exon 1B is indicated. The 1A promoter is predominantly utilized in bone cells;

FIG. 4A provides a map of the BMP-4 1A 5'-flanking-CAT plasmid and promoter activity in FRC cells. The 2.6 kb EcoR1 and Xba fragment, 1.3 kb Pst fragment, 0.5 kb SphI and Pst fragment, and 0.25 kb PCR fragment were inserted into pBLCAT3. The closed box indicates the non-coding exon 1A. The CAT box represents the CAT reporter gene. The values represent percentages of CAT activity expressed by pCAT-2.6 set at 100%. The values represent the average of four independent assays;

FIG. 4B provides an autoradiogram of CAT assays using FRC cells transfected with BMP-4 1A 5'-flanking-CAT plasmids identified in FIG. 4A;

FIG. 5 portrays the nucleotide sequence of the mouse BMP-2 gene 5'-flanking region from -2736 to +139 (SEQ. ID NO. 4). The transcription start site is denoted by +1;

FIG. 6A depicts an autoradiogram showing products of a primer extension assay for determination of the transcription start site of the BMP2 gene, separated on a 8% denaturing urea-polyacrylamide gel, in which Lane 1: Total RNA from fetal rat calvarial osteoblast cells, and Lane 2: Control lane with 10 .mu.g of yeast tRNA. All RNA samples were primed with a .sup.32 P-labeled oligonucleotide from exon 1 of the mouse BMP2 gene, as indicated in FIG. 6B. Lane M: .sup.32 P-labeled MspI digested .lambda. phage DNA, containing DNA fragments spanning from 623 bp to 15 bp (size marker);

FIG. 6B provides a schematic representation of the primer extension assay. The primer used is a 18 mer synthetic oligonucleotide, 5'-CCCGGCAAGTTCAAGAAG-3' (SEQ. ID NO. 5);

FIG. 7 provides a diagram of selected BMP-2 promoter--luciferase reporter constructs. BMP-2 5'-flanking sequences are designated by hatched boxes (.quadrature.) and luciferase cDNA is designated by the filled box (.box-solid.). Base +114 denotes the 3' end of the BMP-2 gene in all the constructs;

FIG. 8 displays the luciferase enzyme activity for the BMP-2 gene-LUC constructs (shown in FIG. 7) transfected in primary fetal rat calvarial osteoblasts (A), HeLa cells (B) and ROS 17/2.8 osteoblasts (C). The luciferase activity has been normalized to .beta.-galactosidase activity in the cell lysates;

FIG. 9A-F depicts the DNA sequence of the mouse BMP-2 promoter and gene (SEQ. ID NO. 6); and

FIG. 10A-D depicts the DNA sequence of the mouse BMP-4 promoter and gene (SEQ. ID NO. 7).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A cell-based assay technique for identifying and evaluating compounds which stimulate the growth of bone is provided, comprising culturing a host cell line comprising an expression vector comprising a DNA sequence encoding a promoter region of at least one bone morphogenetic protein operatively linked to a reporter gene encoding an assayable product under conditions which permit expression of said assayable product, contacting the cultured cell line with at least one compound suspected of possessing osteogenic activity, and identifying osteogenic agents by their ability to modulate the expression of the reporter gene and thereby increase the production of the assayable product.

The present invention is distinguished from other techniques for identifying bone-active compounds, as it specifically identifies chemical compounds, agents, factors or other substances which stimulate bone cells to produce the bone growth factors in the bone morphogenetic protein (BMP) family (hereinafter "osteogenic agents"). These osteogenic agents are identified by their capacity to increase the activity of the promoters of genes of members of the BMP family and other bone growth factors which are normally produced by bone cells, and other cells including cartilage cells, tumor cells and prostatic cells. When patients are treated with such chemical compounds, the relevant BMP will be produced by bone cells and then be available locally in bone to enhance bone growth or bone healing. Such compounds identified by this assay technique will be used for the treatment of osteoporosis, segmental bone defects, fracture repair, prosthesis fixation or any disease associated with bone loss.

Compounds that inhibit bone morphogenetic protein expression in bone or cartilage may also be useful in clinical situations of excess bone formation which occurs in such diseases as osteoblastic metastases or osteosclerosis of any cause. Such compounds can also be identified in accordance with the present invention.

The promoters of the genes for BMP-4 and BMP-2 are complex promoters which can be linked to reporter genes, such as e.g. the firefly luciferase gene. When these hybrid genes (for example, bone cell BMP-4 promoter or bone cell BMP-2 promoter and firefly luciferase, chloramphenicol acetyl transferase (CAT) cDNAs, or cDNA's for other reporter genes such as .beta.-galactosidase, green fluorescent protein, human growth hormone, alkaline phosphatase, .beta.-glucuronidase, and the like) are transfected into bone cells, osteogenic agents which activate the BMP-4 or BMP-2 promoters can be identified by their capacity in vitro to increase luciferase activity in cell lysates after cell culture with the agent.

Sequence data on small fragments of the 5'-flanking region of the BMP-4 gene have been published (Chen et al, 1993; Kurihara et al, 1993), but the promoter has not been previously identified or isolated, and methods for regulating transcription have not been shown. The present invention isolates the promoters for the BMP genes and utilizes these promoters in cultured bone cells so that agents could be identified which specifically increase BMP-2 or BMP-4 production locally in bone. Since it is known that the BMPs are produced by bone cells, a method for enhancing their production specifically in bone should avoid systemic toxicity. This benefit is obtained by utilizing the unique tissue specific promoters for the BMPs which are provided herein, and then using these gene promoters to identify agents which enhance their activity in bone cells.

By utilizing the disclosure provided herein, other promoters can be obtained from additional bone morphogenetic proteins such as BMP-3, BMP-5, BMP-6, and BMP-7, to provide comparable benefits to the promoters herein specifically described.

In addition, the present invention contemplates the use of promoters from additional growth factors in osteoblastic cells. Included are additional bone morphogenetic proteins, as well as fibroblast growth factors (e.g. FGF-1, FGF-2, and FGF-7), transforming growth factors .beta.-1, .beta.-2, and .beta.-3, insulin-like growth factor-1, insulin-like growth factor-2, platelet-derived growth factor, and the like. Such promoters will readily be utilized in the present invention to provide comparable benefits.

The cells which can be utilized in the present invention include primary cultures of fetal rat calvarial osteoblasts, established bone cell lines available commercially (MC3T3-E1 cells, MG-63 cells, U2OS cells, UMR106 cells, ROS 17/2.8 cells, SaOS2 cells, and the like as provided in the catalog from the American Type Culture Collection (ATCC)), and bone cell lines established from transgenic mice, as well as other cell lines capable of serving as hosts for the present vectors and systems. In addition, a number of tumor cell lines also express BMPs, including the prostate cancer cell lines PC3, LNCAP, and DUI145, as well as the human cancer cell line HeLa. Thus, any of a number of cell lines will find use in the present invention and the choice of an appropriate cell line will be a matter of choice for a particular embodiment.

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

EXPERIMENTAL

In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); mM (millimolar); .mu.M (micromolar); N (Normal); mol (moles); mmol (millimoles); .mu.mol (micromoles); nmol (nanomoles); kg (kilograms); gm (grams); mg (milligrams); .mu.g (micrograms); ng (nanograms); L (liters); ml (milliliters); .mu.l (microliters); vol (volumes); and .degree.C. (degrees Centigrade).

EXAMPLE 1

Description and Characterization of Murine BMP-4 Gene Promoter

(a) Library Screening, Cloning and Sequencing of Gene

A mouse genomic lambda fix II spleen library (Stratagene, La Jolla, Calif.) was screened with a mouse embryo BMP-4 cDNA kindly provided by Dr. B. L. M. Hogan (Vanderbilt University School of Medicine, Nashville, Tenn.). The probe was labeled with [.alpha.-.sup.32 P] dCTP using a random-primer labeling kit from Boehringer-Mannheim (Indianapolis, Ind.). Plaque lift filters were hybridized overnight in 6.times. SSC, 5.times. Denhardt's, 0.5% SDS containing 200.mu.g/ml sonicated salmon sperm DNA, 10 .mu.g/ml Poly A and 10 .mu.g/ml t-RNA at 68.degree. C. The filters were washed at 55.degree. C. for 20 min, twice in 2.times. SSC, 0.1% SDS buffer, once in 0.5.times. SSC, 0.1% SDS. The isolated phage DNA clones were analyzed according to standard procedures (Sambrook et al., 1989).

Fragments from positive clones were subcloned into pBluescript vectors (Stratagene, La Jolla, Calif.) and sequenced in both directions using the Sequenase dideoxynucleotide chain termination sequencing kit (U.S. Biochemical Corp., Cleveland, Ohio).

Three clones were isolated from 2.times.10.sup.6 plaques of mouse spleen 129 genomic library using full length coding region mouse embryo BMP-4 cDNA probe (B. Hogan, Vanderbilt University, Nashville, Tenn). One 19 kb clone contained 5 exons and .about.6 kb 5'-flanking region and a .about.7 kb 3'-flanking region, as shown in FIG. 1A. The 7 kb transcription unit and the 5'-flanking region of the mouse BMP-4 gene were sequenced (FIG. 10).

The nucleotide sequence of selected portions of mouse BMP-4 and the deduced amino acid sequence of the coding exons (408 residues; SEQ. ID NO. 2) is shown in FIG. 1B. Primers used in the RT-PCR experiments described below are indicated in this Figure.

FIG. 1C shows the DNA sequence of 2372 bp of the 5'-flanking region and the candidate DNA response elements upstream of exon 1A. Primers used in primer extensions are also shown in FIGS. 1B and 1C.

(b) Primer Extension Mapping of the Transcriptional Start-Site of the Mouse BMP-4 Gene

The transcriptional start-sites were mapped by primer extension using the synthetic oligonucleotide primer A 5'-CGGATGCCGAACTCACCTA-3' (SEQ. ID NO. 8), corresponding to the complement of nucleotides +114 to +96 in the exon 1A sequence and the oligonucleotide primer B1 5'-CTACAAACCCGAGAACAG-3' (SEQ. ID NO. 9), corresponding to the complement of nucleotides +30 to +13 of the exon 1B sequence. Total RNA from fetal rat calvarial (FRC) cells and 9.5 day mouse embryo (gift of B. Hogan, Vanderbilt University) was used with both primers. The primer extension assay was carried out using the primer extension kit from Promega (Madison, Wis.). The annealing reactions were, however, carried out at 60.degree. C. in a water bath for 1 hr. The products were then electrophoresed on 8% denaturing-urea polyacrylamide gels and autoradiographed.

One additional oligonucleotide primer B2 5'-CCCGGCACGAAAGGAGAC-3' (SEQ. ID NO. 10), corresponding to the complement of nucleotide sequence +69 to +52 of exon 1B, was also utilized in primer extension reactions with FRC and mouse embryo RNAs.

1. Evidence for utilization of two alternate exon 1 sequences for the BMP-4 gene

Several BMP-4 cDNAs were sequenced from prostate cancer cell line PC-3 and from primary FRC cells. Four independent FRC cell BMP-4 cDNAs all contained exon 1A. However, the human prostate carcinoma cell line (PC-3) cDNA contained an apparently unique exon 1B sequence spliced to exon 2 (Chen et al, 1993). A double-stranded oligonucleotide probe (70 bp) to exon 1B was synthesized based on the human PC-3 exon 1B sequence. This exon 1B probe was then used to identify the exon 1B region in the mouse genomic BMP-4 clone. The candidate exon 1B is 1696 bp downstream from the 3' end of exon 1A.

2 . Primer extension analysis

Primer extension analysis was performed to map the mouse BMP-4 gene transcription start sites. Primer A, an oligonucleotide from exon 1A, was used and two oligonucleotides from exon 1B. Total RNA was utilized both from mouse embryo and FRC cells. As shown in FIG. 2, a major extended fragment from primer A was obtained in both mouse embryo and FRC cell total RNAs, which migrates at 115 bp. The extended 5'-end of the 115 bp fragment represents the major transcription start site for 1A-containing transcripts. The size of this 5' non-coding exon 1A is 306 bp. A major extended fragment from the complement of primer B1 (exon 1B) was not detected using both mouse embryo and FRC cell total RNAs. One other primer from exon 1B also gave negative results, suggesting that in 9.5 day mouse embryo and FRC cells, the exon 1B-containing transcripts were not detectable, which suggests that transcripts containing exon 1B are less abundant in these cells and tissues than transcripts containing exon 1A. All primer extensions were carried out after annealing of primers at high stringency. Lower stringency annealing with 1B primers gave extended products not associated with BMP-4 mRNA.

Four FRC BMP-4 cDNA were sequenced and found to contain exon 1A sequences spliced to exon 2. The human U2OS BMP-4 cDNA sequence also contains exon 1A (Wozney et al, 1988). This suggests the BMP-4 gene sequences upstream of exon 1A are used primarily in bone cells.

To test whether the BMP-4 1B promoter is utilized at all in FRC cells, oligonucleotide primers were designed to ascertain whether spliced 1B-2-3 exon products and 1A-2-3 exon (control) products could be obtained by a more sensitive RT-PCR technique using FRC poly(A.sup.+)-RNA. The 3' primer was in exon 3 (FIG. 1B--primer 3) and the 5' primers were either in exon 1A (primer 1) or exon 1B (primer 2).

The RT-PCR products were cloned and sequenced. A photograph and diagram of the products obtained are presented in FIG. 3A and B. Both 1A-2-3 and 1B-2-3 products were obtained. The results indicate FRC osteoblasts produce transcripts with either a 1A exon or a 1B exon, but not both. This suggests that the intron region between 1A and 1B exons could contain regulatory response elements under certain conditions. Of the 1B-2-3 RT-PCR products obtained from FRC osteoblasts, two products were obtained with different 3' splice sites for the exon 1B. By comparison with the genomic DNA, both 3' ends of the two exon 1Bs have reasonable 5' splice consensus sequences, consistent with an alternate splicing pattern obtained for the 1B-2-3 RT-PCR products. Most importantly, no 1A-1B-2-3 RT-PCR splice products of the BMP-4 gene were obtained. Thus, 1B does not appear to be an alternatively spliced 5'-non-coding exon. By quantitative RT-PCR, it was shown that 1A transcripts are 10 to 15.times. more abundant in primary bone cells.

The technique of performing RT-PCR will be described. First-strand cDNA was synthesized from 10 .mu.g FRC cell poly(A+)-RNA with an 18 mer dT primer using Superscript.TM. reverse transcriptase (Gibco BRL) in a total volume of 20 .mu.l. The cDNA was then used as a template for PCR with two sets of synthesized primers. As shown in FIG. 1B, primer 1 (5'-GAAGGCAAGAGCGCGAGG-3) (SEQ. ID NO.11), corresponding to a 3' region of exon 1A and primer 3 (5'-CCCGGTCTCAGGTATCA-3') (SEQ. ID NO. 12), corresponding to a 5' region of exon 3 were used to generate exon 1A-2-3 spliced PCR product. Primer 2 (5'-CAGGCCGAAAGCTGTTC-3') (SEQ. ID NO. 13), corresponding to a 3' region (+2 to +18) of exon 1B, and primer 3 were used to generate exon 1B-2-3 spliced PCR products. GeneAmp PCR kit was used according to the manufacturer's procedure (Perkin-Elmer/Cetus, Norwalk, Conn.). Each cycle consisted of a denaturation step (94.degree. C. for 1 min), an annealing step (59.degree. C. for 2 min) and an elongation step (72.degree. C. for 1 min). The PCR products were analysed by agarose gel electrophoresis for size determination. The products were subcloned into pCR II vector using TA cloning kit (InVitrogen, San Diego, Calif.). The inserts were sequenced in both directions with a sequencing kit from U.S. Biochemical (Cleveland, Ohio).

Northern analysis demonstrated that the single 1.8 kb BMP-4 transcript detected in FRC cells during bone cell differentiation hybridizes to both a pure 1A exon probe and a 2-4 exons probe. The ratio of the 1A to 2-4 signal is constant through the changing levels of BMP-4 expression during differentiation. Using a 1B exon probe no detectable hybridization to the BMP-4 exon 2-4 1.8 kb signal was observed. This again indicates that 1A containing transcripts predominate in bone cells, although 1B transcripts can be detected by the more sensitive PCR method. By quantitative PCR it was shown that 1A transcripts are 10-15.times. more abundant than 1B in FRC cells.

(d) BMP-4 Promoter 1A Plasmid Construction and Transfection, and Detection of Promoter Activity in Osteoblasts

Three BMP-4 1A promoter/plasmids were constructed by excising fragments from the 5' flanking region of the mouse BMP-4 gene and cloning into pBL3CAT expression vectors (Luckow and Schutz, 1987). The pCAT-2.6 plasmid was the pBLCAT3 vector with a 2.6 kb EcoR1 and Xba I fragment (-2372/+258) of the BMP-4 gene. The pCAT-1.3 plasmid was similarly generated from a 1.3 kb Pst fragment (-1144/+212). The pCAT-0.5 plasmid was made from a 0.5 kb SphI and Pst fragment (-260/+212). Both the pCAT-1.3 and the pCAT-0.5 plasmids have 212 bp of exon 1A non-coding region. An additional promoter/plasmid was created from a PCR amplified product, corresponding to the 240 bp sequence between nucleotides -25 and +212, and referred to as the pCAT-0.24. The amplified fragment was first cloned into pCR II vector using TA cloning kit (InVitrogen, San Diego, Calif.) and then the fragment was released with Hind III and Xho I, and religated into pBL3CAT. Correct orientation of all inserts with respect to the CAT vector was verified by DNA sequencing.

The cells used for transient transfection studies were isolated from 19 day-old fetal rat calvariae by sequential digestion with trypsin and collagenase, as described by Bellows et al, (1986) and Harris et al, (1994). In brief, the calvarial bone were surgically removed and cleaned by washing in .alpha. minimal essential media (.alpha.MEM) containing 10% V/V fetal calf serum (FCS) and antibiotics. The bones were minced with scissors and were transferred to 35 mm tissue culture dish containing 5 ml of sterile bacterial collagenase (0.1%) and trypsin (0.05%). This was then incubated at 37.degree. C. for 20 min. The cells released at this time were collected and immediately mixed with an equal volume of FCS to inactivate trypsin. This procedure is repeated 6 times to release cells at 20 min intervals. Cells released from 3rd, 4th, 5th and 6th digestion (enriched for osteoblasts) were combined and the cells are collected by centrifugation at 400.times.g for 5 min. The cells were then plated in .alpha.MEM containing 10% FCS and antibiotics and were grown to confluency (2-3 days). At this stage the cells were plated for transfection in 60 mm tissue culture dishes at a cell density of 5.times.10.sup.5 cells per dish. These primary osteoblast cultures are capable of self-organizing into bone-like structure in prolonged cultures (Bellows et al, 1986; Harris et al, 1994). HeLa, ROS 17/2.8, and CV-1 cells were purchased from the ATCC.

The isolated FRC cells, enriched for the osteoblast phenotype, were used as recipient cells for transient transfection assays. BMP-4 mRNA is modulated in these cells in a transient fashion during prolonged cultured (Harris et al, 1994b). The technique of electroporation was used for DNA transfection (Potter, 1988; van den Hoff et al, 1992). After electroporation, the cells were divided into aliquots, replated in 100 mm diameter culture dishes and cultured for 48 hrs in modified Eagle's minimal essential media (MEM, GIBCO, Grand Island, N.Y.) with 10% fetal calf serum (FCS). The extracts were assayed for CAT activity according to the method described by Gorman (1988) and CAT activity was normalized by .beta.-galactosidase assay according to the method of Rouet et al(1992).

After 48 hrs of transfection with various BMP-4-CAT reporter gene plasmid constructs, the cells were harvested and the CAT activity was determined. As indicated in FIGS. 4A and 4B, pCAT-0.24 plasmid (-25/+212) has little CAT activity. This plasmid contains -25 to +212 of the 5' non-coding exon 1A and was 3-fold lower that the parent pBL3CAT plasmid. The pCAT-0.5 (-260/+212), pCAT-1.3 (-1144/+212), and pCAT-2.6 (-2372/+258) showed progressive increasing CAT activity when transfected into FRC cells. These data are shown in FIG. 4B. With pCAT-0.5 (-260/+212) there is a 10-fold increase in CAT activity relative to pCAT-0.24 (-25/+212). pCAT-1.3 (-1144/+212) shows a further 6-fold increase and pCAT-2.6 (-2372/+258) shows further 2-fold change over pCAT-1.3 (-1144/+212). Thus the net increase in CAT activity between the pCAT-0.24 (-257/+212) and the pCAT-2.6 (-2372/+258) in FRC cells is approximately 100-fold.

EXAMPLE 2

Description and Characterization of Murine BMP-2 Gene Promoter

(a) Cloning of Mouse BMP-2 Genomic DNA

Genomic clones of the mouse BMP-2 gene were isolated in order to determine the transcriptional regulation of the BMP-2 gene in primary osteoblasts. 5.times.10.sup.6 plaques were screened from a mouse genomic library, B6/CBA, (purchased from Stratagene, San Diego, Calif.) using BMP-2 cDNA as probe. The BMP-2 cDNA clone was isolated from a cDNA library of PC3 prostate cancer cells (Harris et al, 1994). The human BMP-2 probe was a 1.1 kb SmaI fragment containing most of the coding region.

The BMP-2 genomic clones were sequenced by dideoxy chain termination method (Sanger et al, 1977), using deoxyadenosine 5'-[.alpha.[.sup.35 S]thio] triphosphate and Sequenase (United States Biochemical, Cleveland, Ohio). All fragments were sequenced at least twice and overlaps were established using the appropriate oligonucleotide primer. Primers were prepared on an Applied Biosystems Model 392 DNA Synthesizer. Approximately 16 kb of one of these BMP-2 clones was completely sequenced (FIG. 9). Analysis of this sequence showed that the mouse BMP-2 gene contains one noncoding and two coding exons (Feng et al, 1994). Analysis of the 5' flanking sequence showed that the BMP-2 gene does not contain typical TATA or CAAT boxes. However, a number of putative response elements and transcription factor recognition sequences were identified upstream of exon 1 (FIG. 5). The 5'-flanking region is GC rich with several SP-1, AP-1, P53, E-box, homeobox, and AP-2 candidate DNA binding elements.

(b) Analysis of Transcription Start Site for BMP-2 Gene

The transcription start sites for the BMP-2 gene were identified using the primer extension technique. Primer extension was carried out as described (Hall et al., 1993). The primer used was a .sup.32 P-labeled 18 mer oligonucleotide 5'-CCCGGCAATTCAAGAAG-3' (SEQ. ID NO. 5). Total RNA obtained from primary fetal rat calvarial osteoblasts, was used for the primer extension. The results are shown in FIG. 6. The major extension product was 68 bp and was used to estimate the major transcription start site (+1, FIG. 5). These results were confirmed by RNase protection assays.

The BMP-2-LUC constructs (FIG. 7) were designed to contain variable 5' boundaries from BMP-2 5'-flanking sequences spanning the transcription start site (+1). Each construct contained the 3' boundary at +114 in exon 1 (FIG. 6). These constructs were individually transfected into primary cultures of fetal rat calvarial osteoblasts, ROS 17/2.8 osteosarcoma cells, HeLa cells, and CV-1 cells by the calcium-phosphate precipitation technique and the promoter activity for each of these constructs was assayed 24 hrs following transfection by measuring the luciferase enzyme activity for each individual cell lysate. The LUC (luciferase enzyme assay) technique is described below under (f). Plasmid pSV.beta.Gal was co-transfected with each plasmid construct to normalize for the transfection efficiency in each sample. The experiments were repeated at least five times in independent fetal rat calvarial cultures, with each assay done in triplicate. The mean values from a representative experiment are shown in FIG. 8.

(d) Isolation of Primary Fetal Rat Calvarial Osteoblasts for Functional Studies of BMP-2 Gene Promoter

The cells used for transient transfection studies were isolated from 19 day-old fetal rat calvariae by sequential digestion with trypsin and collagenase, as described by Bellows et al., (1986) and Harris et al., (1994). In brief, the calvarial bone were surgically removed and cleaned by washing in .alpha. minimal essential media (.alpha.MEM) containing 10% V/V fetal calf serum (FCS) and antibiotics. The bones were minced with scissors and were transferred to 35 mm tissue culture dish containing 5 ml of sterile bacterial collagenase (0.1%) and trypsin (0.05%). This was then incubated at 37.degree. C. for 20 min. The cells released at this time were collected and immediately mixed with an equal volume of FCS to inactivate trypsin. This procedure was repeated 6 times to release cells at 20 min intervals. Cells released from 3rd, 4th, 5th and 6th digestion (enriched for osteoblasts) were combined and the cells were collected by centrifugation at 400 g for 5 min. The cells were then plated in .alpha.MEM containing 10% FCS and antibiotics and were grown to confluency (2-3 days). At this stage the cells were plated for transfection in 60 mm tissue culture dishes at a cell density of 5.times.10.sup.5 cells per dish. These primary osteoblast cultures are capable of mineralized bone in prolonged cultures (Bellows et al, 1986; Harris et al, 1994). HeLa, ROS 17/2.8, and CV-1 cells were purchased from the ATCC.

(e) Transient Transfection Assay

For transient transfection assay, the primary osteoblast cells were plated at the above mentioned cell density 18-24 hrs prior to transfection. The transfection was carried out using a modified calcium-phosphate precipitation method (Graham & van der Eb 1973; Frost & Williams 1978). The cells were incubated for 4 hrs at 37.degree. C. with 500 .mu.l of a calcium phosphate precipitate of plasmid DNA containing 10 .mu.g of reporter plasmid construct and 1 .mu.g of pSV.beta.Gal (for normalization of transfection efficiency) in 0.15 M CaCl.sub.2 and Hepes buffered saline (21 mM Hepes, 13.5 mM NaCl, 5 mM KCl, 0.7 mM Na.sub.2 HPO.sub.4, 5.5 mM dextrose, pH 7.05-7.1). After the 4 hr incubation period of cells with precipitate, the cells were subjected to a 2 min treatment of 15% glycerol in .alpha.MEM, followed by addition of fresh .alpha.MEM containing insulin, transferrin and selenium (ITS) (Upstate Biotechnology, Lake Placid, N.Y.). The cells were harvested 24 hrs post transfection.

(f) Luciferase and .beta.-galactosidase Assay

Cell lysates were prepared and luciferase enzyme assay was carried out using assay protocols and the assay kit from Promega (Madison, Wis.). Routinely 20 .mu.l of cell lysate was mixed with 100 .mu.l of luciferase assay reagent (270 .mu.M coenzyme A, 470 .mu.M luciferin and 530 .mu.M ATP) and the luciferase activity was measured for 10 sec in a TURNER TD-20e luminometer. The values were normalized with respect to the .beta.-galactosidase enzyme activity, obtained for each experimental sample.

The .beta.-galactosidase enzyme activity was measured in the cell lysate using a 96 well microtiter plate according to Rouet et al (1992). 10-20 .mu.l cell lysate was added to 90-80 .mu.l .beta.-galactosidase reaction buffer containing 88 mM phosphate buffer, pH 7.3, 11 mM KCl, 1 mM MgCl.sub.2, 55 mM .beta. mercaptoethanol, 4.4 mM chlorophenol red .beta.-D-galactopyranoside (Boehringer-Mannheim Corp., Indianapolis, Ind.). The reaction mixture was incubated at 37.degree. C. for 30-60 min, depending on transfection efficiency, and the samples were read with an ELISA plate reader at 600 nm.

(g) Plasmid Construction

The luciferase basic plasmid (pGL basic) was the vector used for all constructs (purchased from Promega, Madison, Wis.). Different lengths of DNA fragments from the BMP-2 5'-flanking region were cloned at the multiple cloning sites of this plasmid, which is upstream of the firefly luciferase cDNA. The BMP-2 DNA fragments were isolated either by using available restriction enzyme sites (constructs -196/+114, -876/+114, -1995/+114, -2483/+114, and -2736/+114) or by polymerase chain reaction using specific oligonucleotide primers (constructs -23/+114, -123/+114 and +29/+114).

The minimal promoter activity for the BMP-2 gene was identified in the shortest construct containing 23 bp upstream of the transcription start site (-23/+114). No luciferase activity was noted in the construct that did not include the transcription start site (+29/+114). Two other constructs containing increasing lengths of 5' sequences up to -196 bp showed reproducible decreases in promoter activity in fetal rat calvarial osteoblasts and HeLa cells (FIG. 8). The -876/+114 construct showed a 5-fold increase in activity in HeLa cells. The -1995/+114, -2483/+114 and -2736/+114 constructs showed decreased promoter activity when compared to the -876/+114 construct only in HeLa cells (FIG. 8).

In the primary fetal rat calvarial osteoblasts, the 2.6 kb construct (-2483/+114) demonstrated a 2-3-fold increase in luciferase activity over that of the -1995/+114 construct (FIG. 8). These results suggest that one or more positive response regions are present between -196 and -1995 and that the DNA sequence between -1995 and -2483 bp has other positive regulatory elements that could modulate BMP-2 transcription. The largest 2.9 kb construct (-2736/+114) repeatedly demonstrated a 20-50% decrease in promoter activity compared to the -2483/+114 construct, in these primary fetal rat calvarial osteoblasts (FIG. 8).

In ROS 17/2.8 osteosarcoma cells, the BMP-2 promoter activity was consistently higher than either the primary fetal rat calvarial osteoblasts or HeLa cells (FIG. 8). All of the deletion constructs showed similar promoter activity in ROS 17/2.8 osteosarcoma cells. The transformed state in ROS 17/2.8 cells may be responsible for the marked expression of the BMP-2 gene. ROS 17/2.8 cells represent a well differentiated osteosarcoma and they produce high levels of BMP-2 mRNA. They form tumors in nude mice with bone-like material in the tumor (Majeska et al, 1978; Majeska et al, 1980).

(h) Specificity of the BMP-2 Promoter

To analyze the activity of the BMP-2 promoter in cell types not expressing BMP-2 mRNA, BMP-2 promoter constructs were transfected into CV-1 cells (monkey kidney cells). The BMP-2 promoter activity was found to be very low for all constructs. This suggests that this region of the BMP-2 promoter is functional only in cells such as primary fetal rat calvarial osteoblasts, HeLa and ROS 17/2.8 that express endogenous BMP-2 mRNA (Anderson & Coulter 1968). CV-1 cells do not express BMP-2 mRNA. The BMP-2 promoter is likely active in other cell types that express BMP-2, such as prostate cells and chondrocytes, although regulation of transcription may be different in these cells.

EXAMPLE 3

Use of Plasmid Constructs Containing BMP Promoters With Reporter Genes to Identify Osteogenic Agents

Plasmid constructs containing BMP promoters with reporter genes have been transfected into osteoblastic cells. The cells which have been utilized include primary cultures of fetal rat calvarial osteoblasts, cell lines obtained as gifts or commercially (MC3T3-E12 cells, MG-63 cells, U2OS cells, UMR106 cells, ROS 17/2.8 cells, SaOS2 cells, and the like as provided in the catalog from the ATCC) and bone and cartilage cell lines established from transgenic mice. The bone cells are transfected transiently or stably with the plasmid constructs, exposed to the chemical compound, agent or factor to be tested for 48 hours, and then luciferase or CAT activity is measured in the cell lysates.

Regulation of expression of the growth factor is assessed by culturing bone cells in .alpha.MEM medium with 10% fetal calf serum and 1% penicillin/streptomycin and 1% glutamine. The cells are placed in microtiter plates at a cell density of 5.times.10.sup.3 cells/100 .mu.l/well. The cells are allowed to adhere and then incubated at 37.degree. C. at 5% CO.sub.2 for 24 hours and then the media is removed and replaced with 50 .mu.l .alpha.MEM and 4% fetal calf serum. 50 .mu.l aliquots containing the compound or factor to be tested in 0.1% BSA solution is added to each well. The final volume is 100 .mu.l and the final serum concentration is 2% fetal calf serum. Recombinant rat BMP-2 expressed in Chinese hamster ovarian cells is used as a positive control.

The treated cells are incubated at 37.degree. C. at 5% CO.sub.2 for 48 hours. The media is then removed and the cells are rinsed 3 times with phosphate buffered saline (PBS). Excess PBS is removed from the wells and 100 .mu.l of cell culture lysing reagent (Promega #E153A) is added to each well. After 10 minutes, 10 .mu.l of the cell lysate is added to a 96-well white luminometric plate (Dynatech Labs #07100) containing 100 .mu.l luciferase assay buffer with substrate (Promega #E152A). The luciferase activity is read using a Dynatech ML2250 automated 96-well luminometer. The data is expressed as either picograms of luciferase activity per well or picograms of luciferase activity per .mu.g protein.

EXAMPLE 4

Demonstration That Bone Cells Transfected With BMP Promoters Can Be Used To Screen for Osteogenic Agents

To demonstrate that the present invention is useful in evaluating potential osteogenic agents, a random array of chemical compounds from a chemical library obtained commercially was screened. It was found that approximately 1 in 100 such compounds screened produces a positive response in the present assay system compared with the positive control, recombinant BMP-2, which is known to enhance BMP-2 transcription. Compounds identified from the random library were subjected to detailed dose-response curves, to demonstrate that they enhance BMP messenger RNA expression, and that they enhance other biological effects in vitro, such as expression of structural proteins including osteocalcin, osteopontin and alkaline phosphatase, and enhance bone nodule formation in prolonged primary cultures of calvarial rodent osteoblasts.

Compounds identified in this way can be tested for their capacity to stimulate bone formation in vivo in mice. To demonstrate this, the compound can be injected locally into subcutaneous tissue over the calvarium of normal mice and then the bone changes are followed histologically. It has been found that certain compounds identified by the present invention stimulate the formation of new bone in this in vivo assay system.

The effects of compounds are tested in ICR Swiss mice, aged 4-6 weeks and weighing 13-26 g. The compound at 20 mg/kg or vehicle alone (100 .mu.l of 5% DMSO and phosphate-buffered 0.9% saline) are injected three times daily for 7 days. The injections are given into the subcutaneous tissues overlying the right side of the calvaria of five mice in each treatment group in each experiment.

Mice are killed by ether inhalation on day 14, i.e. 7 days after the last injection of compound. After fixation in 10% phosphate-buffered formalin, the calvariae are examined. The occipital bone is removed by cutting immediately behind and parallel to the lambdoid suture, and the frontal bone is removed by cutting anterior to the coronal suture using a scalpel blade. The bones are then bisected through the coronal plane and the 3- to 4 mm strips of bone are decalcified in 14% EDTA, dehydrated in graded alcohols, and embedded in paraffin. Four 3 .mu.m thick nonconsecutive step sections are cut from each specimen and stained using hematoxylin and eosin.

Two representative sections from the posterior calvarial strips are used. Histological measurements are carried out using a digitizing tablet and the Osteomeasure image analysis system (Osteometrics Inc., Atlanta, Ga.) on the injected and noninjected sides of the calvariae in a standard length of bone between the sagittal suture and the muscle insertion at the lateral border of each bone. Measurements consist of 1) Total bone area (i.e., bone and marrow between inner and outer periosteal surfaces); 2) Area of new woven bone formed on the outer calvarial surface; 3) The extent of osteoblast lined surface on the outer calvarial surface; 4) The area of the outer periosteum; and 5) The length of calvarial surface. From these measurements, the mean width of new bone and periosteum and the percentage of surface lined by osteoblasts on the outer calvarial surface, can be determined.

By reference to the above disclosure and examples, it is seen that the present invention provides a new cell-based assay for identifying and evaluating compounds which stimulate the growth of bone. Also provided in accordance with the present invention are promoter regions of bone morphogenetic protein genes, and a system for identifying osteogenic agents utilizing such promoters operatively linked to reporter genes in expression vectors.

The present invention provides the means to specifically identify osteogenic agents which stimulate bone cells to produce bone growth factors in the bone morphogenetic protein family. These osteogenic agents are shown to be useful to increase the activity of the promoters of genes of members of the BMP family and other bone growth factors normally produced by bone cells.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application are specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art in light of the teaching of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Anderson, H. C. and P. R. Coulter (1968) Fed. Proc. 27, 475.

Bellows, C. G., J. E. Aubin, J. N. M. Heersche and M. E. Antosz (1986) Mineralized bone nodules formed in vitro from enzymatically released rat calvarial cell populations. Calcif. Tissue Int. 38, 143-154.

Chen, D., J. Q. Feng, M. Feng, M. A. Harris, G. R. Mundy and S. E. Harris (1993) Biochim Biophys Acta 1174, 289-292.

Feng, J. Q., M. A. Harris, N. Ghosh-Choudhury, M. Feng, G. R. Mundy and S. E. Harris (1994) Biochem. Biophys. Acta 1218, 221-224.

Frost, E. and J. Williams (1978) Virology 91, 39-50.

Gorman, C. (1988) in DNA Cloning, A Practical Approach (Gover, D. M., ed) Vol. II, pp. 157-158, IRL Press, Oxford, England.

Graham, F. L., and A. J. van der Eb (1973) Virology 52, 456-467.

Hall, J. A., M. A. Harris, R. Intres, and S. E. Harris (1993) J Cell Biochem 51, 116-127.

Harris, S. E., L. F. Bonewald, M. A. Harris, M. Sabatini, S. Dallas, J. Feng, N. Ghosh-Choudhury, J. Wozney and G. R. Mundy (1994) Effects of TGF.beta. on bone nodule formation and expression of bone morphogenetic protein-2, osteocalcin, osteopontin, alkaline phosphatase and Type I collagen mRNA in prolonged cultures of fetal rat calvarial osteoblasts. J Bone Miner Res 9, 855-863.

Harris, S. E., M. Sabatini, M. A. Harris, J. Q. Feng, J. Wozney and G. R. Mundy (1994) Expression of bone morphogenetic protein messenger RNA in prolonged cultures of fetal rat calvarial cells. J Bone Min Res 9, 389-394.

Harris, S. E., M. Harris, M. Mahy, J. Wozney, J. Feng and G. R. Mundy (1994) Expression of bone morphogenetic proteins by normal rat and human prostate and prostate cancer cells. The Prostate 24, 204-211.

Kurihara, T., K. Kitamura, K. Takaoka, H. Nakazato (1993) Murine bone morphogenetic protein-4 gene: existence of multiple promoters and exons for the 5'-untranslated region. Biochem Biophys Res Commun 192, 1049-1056.

Luckow, B. and G. Schutz (1987) Nucleic Acids Res. 15, 5490.

Majeska, R. J., S. B. Rodan and G. A. Rodan (1978) Maintenance of parathyroid hormone response in clonal rat osteosarcoma lines. Exp Cell Res 111, 465-468.

Majeska, R. J., S. B. Rodan and G. A. Rodan (1980) Parathyroid hormone responsive clonal cell lines from rat osteosarcoma. Endocrinology 107, 1494-1503.

Potter, H. (1988) Anal Biochem 174, 361-373.

Rouet, P., G. Raguenez and J-P Salier (1992) Biotechniques 13, 700-701.

Sambrook, J., E. F. Fritsch and T. Maniatis (1989) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Sanger, F., S. G. Nicklen and A. R. Coulson (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467.

Stein, G. S., J. B. Lian and T. A. Owen (1990) Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. FASEB J 4, 3111-3123.

Urist, M. R. (1965) Bone: Formation by autoinduction. Science 150, 893.

van den Hoff, M. J. B., A. F. M. Moorman, and W. H. Lamers (1992) Nucleic Acids Res. 20, 2902.

Wozney, J. M., V. Rosen, A. J. Celeste, L. M. Mitsock, M. J. Whitters, R. W. Kriz, R. M. Hewick and E. A. Wange (1988) Novel regulators of bone formation: Molecular clones and activities. Science 242, 1528-1534.

Wozney, J. M. (1992) The bone morphogenetic protein family and osteogenesis. Mol Reprod Dev 32, 160-167.

Wozney, J. M. and V. Rosen (1993) Bone morphogenetic proteins. In: Physiology and Pharmacology of Bone (edited by Mundy G. R., Martin T. J.). Springer-Verlag, Chapter 20, 725-743.

Claims

We claim:

1. An isolated DNA molecule which comprises the nucleotide sequence at positions -2372 to +316 depicted in FIG. 1C (SEQ. ID NO. 3), or a portion thereof which is effective as a promoter.

2. A recombinant expression vector comprising the portion of the DNA molecule of claim 1 which is effective as a promoter.

3. The recombinant expression vector of claim 2 further comprising a nucleotide sequence encoding an assayable product operatively linked to said portion effective as a promoter.

4. The recombinant expression vector of claim 3 wherein said assayable product is firefly luciferase, chloramphenicol acetyl transferase (CAT), .beta.-galactosidase, green fluorescent protein (GFP), human growth hormone, alkaline phosphatase or .beta.-glucuronidase.

5. A system for identifying osteogenic agents comprising: host cells or a cell line modified to contain the expression vector of claim 3; and means for detecting said assayable product produced in response to exposure to an osteogenic agent.

6. The system of claim 5 wherein said assayable product is firefly luciferase, chloramphenicol acetyl transferase (CAT), .beta.-galactosidase, green fluorescent protein (GFP), human growth hormone, alkaline phosphatase or .beta.-glucuronidase.

7. Recombinant host cells or a recombinant host cell line modified to contain the expression vector of claim 3.

8. A method for identifying an osteogenic agent comprising contacting the cells or cell line of claim 7 with at least one compound suspected of possessing osteogenic activity; and measuring the production of said assayable product in the presence and absence of said compound; whereby a compound which results in an increase in production of said assayable product in its presence as opposed to its absence is identified as an osteogenic agent.

9. The method of claim 8 wherein said assayable product is firefly luciferase, chloramphenicol acetyl transferase (CAT), .beta.-galactosidase, green fluorescent protein (GFP), human growth hormone, alkaline phosphatase or .beta.-glucuronidase.

10. An isolated DNA molecule which comprises the nucleotide sequence at positions -2736 to +139 depicted in FIG. 5 (SEQ. ID NO. 4), or a portion thereof which is effective as a promoter.

11. A recombinant expression vector comprising the portion of the DNA molecule of claim 10 which is effective as a promoter.

12. The recombinant expression vector of claim 11 further comprising a nucleotide sequence encoding an assayable product operatively linked to said portion effective as a promoter.

13. The recombinant expression vector of claim 12 wherein said assayable product is firefly luciferase, chloramphenicol acetyl transferase (CAT), .beta.-galactosidase, green fluorescent protein (GFP), human growth hormone, alkaline phosphatase or .beta.-glucuronidase.

14. A system for identifying osteogenic agents comprising: host cells or a cell line modified to contain the expression vector of claim 12, and means for detecting said assayable product produced in response to exposure to an osteogenic agent.

15. The system of claim 14 wherein said assayable product is firefly luciferase, chloramphenicol acetyl transferase (CAT), .beta.-galactosidase, green fluorescent protein (GFP), human growth hormone, alkaline phosphatase or .beta.-glucuronidase.

16. Recombinant host cells or a recombinant host cell line modified to contain the expression vector of claim 12.

17. A method for identifying an osteogenic agent comprising contacting the cells or cell line of claim 16 with at least one compound suspected of possessing osteogenic activity; and measuring the production of said assayable product in the presence and absence of said compound; whereby a compound which results in an increase in production of said assayable product in its presence as opposed to its absence is identified as an osteogenic agent.

18. The method of claim 17 wherein said assayable product is firefly luciferase, chloramphenicol acetyl transferase (CAT), .beta.-galactosidase, green fluorescent protein (GFP), human growth hormone, alkaline phosphatase or .beta.-glucuronidase.

Patent Citations (5)

Patent	Date	Inventor
US5166058	1992-11-01	Wang et al.
US5266683	1993-11-01	Oppermann et al.
US5491084	1996-02-01	Chalfie et al.
EPX629697	1994-12-01
WOXWO92/13091	1992-08-01

Non-Patent Literature (28)

Alam et al., "Reporter genes: Application to the study of mammalian gene transcription", Anal. Biochm. 188(2): 245-254, Aug. 1990.
Ghosh-Choudhury, N. et al., "Expression of the BMP 2 Gene during Bone Cell Differentiation," Critical Reviews in Eukaryotic Gene Expression (1994) 4(2&3):345-355.
Harris, S.E. et al., "Retinoid Regulation of Bone Morphogenetic Protein 4 (BMP 4 or DVR 4): Analysis of the Mouse BMP 4 Gene Promoter by Transfection Into Primary Cultures of Fetal Rat Calvariae (FC) osteoblasts," J Cell Biochem Suppl C (17 Part A) (1993) 159.
Harris, S.E. et al., "Development of Osteoblast Cell Lines from Transgenic Mice Containing Bone Morphogenic Protein 2 (BMP2) Promoter-T-Antigen Constructs: Analysis of BMP 2 Retinoic Acid and 1,25(OH).sub.2 Vitamin D Response Regions in the BMP 2, Promoter in the Context of Chromatin Structure," J Cell Biochem Suppl. C (18B) (1994).
Anderson, H.C. et al., "Bone-Inducing Capability of Cultured Human Cells (FL and HELA) Compared to that of Various Types of Injury," Fed. Proceed. (1968) 27:475.
Bellows, C.G. et al., "Mineralized Bone Nodules Formed In Vitro from Enzymatically Released Rat Calvaria Cell Populations," Calcif Tissue Int (1986) 38:143-154.
Chen, D. et al., "Cloning and sequence of bone morphogenetic protein 4 cDNA from fetal rat calvarial cell," Biochimica et Biophysica Acta (1993) 1174:289-292.
Feng, J.Q. et al., "Structure and sequence of mouse bone morphogenetic protein-2 gene (BMP-2): comparison of the structures and promoter regions of BMP-2 and BMP-4 genes," Biochimica et Biophysica Acta (1994) 221-224.
Frost, E. et al., "Mapping Temperature-Sensitive and Host-Range Mutations of Adenovirus Type 5 by marker Rescue," Virology (1978) 91:39-50.
Gorman, C. "DNA Cloning, A Practical Approach" (Gover, D.M., ed) (1988) vol. II, pp. 157-158, IRL Press, Oxford, England.
Graham, F.L. et al., "A New Technique for the Assay of Infectivity of Human Adenovirus 5 DNA," Virology (1973) 52:456-467.
Hall, J.A. et al., "Acidic Fibroblast Growth Factor Gene 5' Non-Coding Exon and Flanking Region From Hamster DDT1 Cells: Identification of the Promoter Region and Transcriptional Regulation by Testosterone and aFGF Protein," Journal of Cellular Biochemistry (1993) 51:116-127.
Harris, S.E. et al., "Expression of Bone Morphogenetic Protein Messenger RNAs by Normal Rat and Human Prostate and Prostate Cancer Cells," The Prostate (1994) 24:204-211.
Harris, S.E. et al., "Effects of Transforming Growth Factor .beta. on Bone Nodule Formation and Expression of Bone Morphogenetic Protein 2, Osteocalcin, Osteopontin, Alkaline Phosphatase, and Type I Collagen mRNA in Long-Term Cultures of Fetal Rat Calvarial Osteoblasts," Journal of Bone and Mineral Research (1994) 9(6):855-863.
Harris, S.E. et al., "Expression of Bone Morphogenetic Protein Messenger RNA in Prolonged Cultures of Fetal Rat Calvarial Cells," Journal of Bone and Mineral Research (1994) 9(3):389-394.
Kurihara, T. et al., "Murine Bone Morphogenetic Protein-4 Gene: Existence of Multiple Promoters and Exons for the 5'-Untranslated Region," Biochemical and Biophysical Research Communications (1993) 192(3):1049-1056.
Luckow, B., "CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements," Nucleic Acids Res (1987) 15:5490.
Majeska, R.J. et al., "Maintenance of parathyroid hormone response in clonal rat osteosarcoma lines," Exp Cell Res (1978) 111:465-467.
Majeska, R.J. et al., "Parathyroid Hormone-Responsive Clonal Cell Lines from Rat Osteosarcoma," Endocrinology (1980) 107(5):1494-1502.
Potter, H., "Electroporation in Biology: Methods, Applications, and Instrumentation," Analytical Biochemistry (1988) 174:361-373.
Sanger, F. et al., "DNA sequencing with chain-terminating inhibitors," Proc Natl Acad Sci USA (1977) 74(12) 5463-5467.
Stein, G.S. et al., "Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation," Cell Growth and Gene Expression (1990) 4:3111-3123.
Urist, M.R., "Bone: Formation by Autoinduction," Science (1965) 150:893-899.
van den Hoff, M.J.B. et al., "Electroporation in `intracellular` buffer increases cell survival," Nucleic Acids Research (1992) 20(11):2902.
Wozney, J.M. et al., "Novel Regulators of Bone Formation: Molecular Clones and Activities," Science (1988) 242:1528-1534.
Wozney, J.M., "The Bone Morphogenetic Protein Family and Osteogenesis," Molecular Reproduction and Development (1992) 32:160-167.
Wozney, J.M. et al., "Bone Morphogenetic Proteins," In: Physiology and Pharmacology of Bone (edited by Mundy, G.R., Martin, T.J.). Springer-Verlag, Chapter 20, (1993) pp. 725-743.
Feng, J.Q. et al., "The Mouse Bone Morphogenetic Protein-4 Gene,"Journal of Biological chemistry,vol. 270, No. 47, Nov. 24, 1995, pp. 28364-28373.