The applicants have constructed a hybrid transgene in which SV40 large T-antigen (Tag) expression was directed by the tartrate-resistant acid phosphatase (TRAP) gene promoter to target Tag to the osteoclast (OCL) in transgenic mice, as a means of developing OCL cell lines. They produced five transgenic TRAP-Tag founder mice that had Tag targeted to the OCL. Transgenic lines then were produced from two of these founder mice. All of these mice had increased OCL, transformed OCL, OCL with mitotic figures and hyperploidy, demonstrating the feasibility of this approach. Several TRAP-Tag animals developed OCL tumors in their marrow and three primary cell cultures were isolated that formed OCL, suggesting that OCL cell lines can be produced from these animals. However, permanent cell lines have not as yet been established, possibly reflecting that additional transforming genes are needed to immortalize OCL. In this application, these previous studies will be extended to (1) establish mice transgenic for bcl-xL, fused to the TRAP promoter (TRAP-bcl-xL), interbreed these mice with TRAP-Tag mice to make mice doubly transgenic for Tag and bcl-xL targeted to the OCL, and continue to further characterize the TRAP-Tag transgenic mice, and attempt to develop and characterize OCL cell lines from these mice; (2) clone the promoter for collagenase Type IV (Col4B), a gene expressed very early in the OCL lineage, characterize the Col4B promoter activity, and confirm by in situ hybridization that levels of Col4B mRNA are markedly increased in OCL versus other bone cells; (3) produce transgenic TRAP-myc and Col4B- ras mice to determine their capacities to develop OCL tumors, characterize these tumors and determine if cell lines can be derived from these tumors, crossbreed TRAP-myc to Col4B-ras or TRAP-Tag or TRAP-bcl- xL mice to produce doubly transgenic mice; and (4) determine the effects of osteotropic factors on OCL cell lines derived from these transgenic mice.
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