The matrix protein of bones and teeth play key roles in the structure and functions of these tissues. Our objective was to study their function and regulation using a combination in vitro and in vivo analysis. The genes we have studied are biglycan (BGN) and bone sialoprotein (BSP) both of which are highly expressed in bones and teeth. Our first approach was to use isolated cells to study gene control at the nuclear level. Such experiments employed cDNA and genomic DNA isolation and cloning as well as extensive DNA gene mapping and sequencing. To understand the mechanisms that control RNA production matrix, we characterized the promoters for these genes by transfecting them into cultured skeletal cells. DNase protection mobility shift assay, UV cross-linking and southwestern blotting were also used to understand the nature of the DNA protein interactions that control BSP and BGN gene expression. Our studies indicated the transcription factors YY1 and c-krox were key in the control of these genes respectively. To study matrix protein function, experiments are underway to create transgenic mice that are null or """"""""knockout"""""""" (not able to make) for specific matrix genes. Our approach is to replace specific genes by a targeting method that relies on homologous recombination in embryonic stem cells. In addition, in parallel, we are creating mice that have a """"""""gain of function"""""""" (make more of a specific gene) using conventional transgenics. It is theorized that the combination of targeted """"""""gene knockout"""""""" and """"""""gain of function"""""""" transgenic animals will provide new insight into the role of matrix proteins in the development and aging of skeletal tissue.
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