During the last grant period, we developed an osteoclast (OCL) precursor cell line by targeting the bcl-XL and SV40 large T antigen genes to cells in the OCL lineage (B/T cells). These cells form high numbers of OCLs (more than 500 times greater than normal marrow) and have been in culture for more than 2 years. This OCL precursor cell line provides us with the unique opportunity to identify genes intrinsic to OCLs whose expression is required for formation of mature OCLs. It is our hypothesis that the sequential high level expression of a small number of genes (novel and known) is critical for commitment of precursors to differentiate to mature OCLs. To test this hypothesis, we will: (1) Assess the importance in OCL formation in vitro of the 4 genes we have recently identified that are overexpressed in mature OCLs compared to their precursors. We will transfect B/T cells treated with RANK ligand and M-CSF or vehicle in the absence of stromal cells with sense or antisense constructs for these genes, to enhance overexpression of these genes or block their effects and determine if OCLs or macrophages form. Based on these assays, a candidate gene that appears most critical to OCL formation will be selected for in vivo studies; (2) Use the tartrate-resistant acid phosphatase (TRAP) promoter to overexpress this candidate gene in cells of the OCL lineage of transgenic mice. We will assess its effects on OCL activity in these overexpressor mice by quantifying OCL number and activity; (3) Disrupt expression of the candidate gene in OCLs and their precursors in mice using the Cre/lox system of gene targeting, and assess the loss of function of this gene in OCLs as follows: (a) Target the Cre gene to cells in the OCL lineage in vivo using the TRAP promoter; (b) Develop mice in which the wild type candidate gene is replaced by an altered form of the gene carrying two loxP sites using gene targeting technology in embryonic stem cells; (c) Interbreed TRAP-Cre transgenic mice to the lox-candidate gene mice to develop mice that lack the candidate gene specifically in OCLs and assess OCL activity in these mice.
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