Our objectives are concerned with the molecular mechanism and requirements for the Immunoglobulin (Ig) Heavy chain Constant Region (CH) gene switch and the regulation of IG gene expression by B cell specific trans-acting factors. More specifically, we plan to continue our molecular studies of successive, C-Gama gene switches in MPC-11 class switch variants. We also describe a novel in vivo selection assay for switch recombinase activities in lymphoid cell lines. Plasmid and retroviral vectors harboring different S region substrates will be introduced into pre B cell lines with well defined CH switching capability. Switch-recombination events will be selected by the loss of a genetic marker (the HSV-Thymidine Kinase gene) placed in between different S regions. In our last specific aim, we will examine the mechanism of regulation of the Ig H chain gene transcriptional enhancer (Igh-E) by trans-acting, tissue specific factors. Once again, we will employ an in vivo selection assay. Here, we will combine a selectable marker (the neomycin resistance gene) placed under Igh-E control and somatic cell hybrid techniques to rescue B cell chromosomes harboring such trans-acting factors. Our strategy will rely on the observation that fibroblasts transformed with a neo gene under Igh-E control do not acquire resistance to neomycin. Therefore we will attempt to rescue neo expression by fusion with a myeloma cell line. Cell hybrids will be selected for with independent genetic markers. If this approach is successful, we will employ this biological assay to isolate the gene(s) encoding such factors from a B cell cDNA library prepared in a mammalian expression vector. This technology has the potential to isolate and characterize genes which regulate H and L chain gene expression.
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