SWITCH MECHANISM FOR GENE EXPRESSION IN TRANSGENIC

THIS PROJECT SEEKS TO GENERATE EUKARYOTIC CELLS AND, ULTIMATELY, TRANSGENIC MOUSE STRAINS THAT EXPRESS EXOGENOUS GENES UNDER TIGHT REGULATORY CONTROL OF A BACTERIAL REPRESSOR. THIS HYBRID EUKARYOTIC/PROKARYOTIC SWITCH WILL GREATLY EXTEND THE USEFULNESS OF EXISTING EUKARYOTIC PROMOTERS AND INCREASE THE POTENTIAL FOR GENERATING USEFUL TRANSGENIC ANIMAL MODELS FOR STUDYING AUTOIMMUNE DISEASES, IMMUNE TOLERANCE, MUTAGENICITY, ONCOGENICITY, CELL DIFFERENTIATION, AND GENE EXPRESSION. IN PHASE I, THE COMPONENTS OF THE PROKARYOTIC LAC OPERON WILL BE TESTED IN STABLY TRANSFECTED MOUSE L CELLS. CELLS EXPRESSING THE LAC REPRESSOR WILL THEN BE STABLY TRANSFECTED WITH CHIMERIC GENE CONSTRUCTS CONTAINING THE LACOPERATOR SEQUENCE. WILD-TYPE AND TIGHT-BINDING REPRESSORS AND OPERATOR SEQUENCES WILL BE TESTED. THE EFFICIENCY OF NUCLEAR TRANSPORT OF THE LAC REPRESSOR CONTAINING A SIGNAL PEPTIDE SEQUENCE WILL BE TESTED BY MEASURING THE NUCLEAR CONCENTRATION OF THE LAC REPRESSOR, AND BOTH THE LEVEL OF REPRESSOR EXPRESSION AND THE NUMBER AND RELATIVE POSITIONING OF OPERATOR SEQUENCES WILL BE TESTED. ALTHOUGH THESE EXPERIMENTS WILL FOCUS ON MAXIMAL REPRESSION FOR GENES REQUIRING LOW LEVELS OF EXPRESSION, CONDITIONS FOR MAXIMAL INDUCTION WILL ALSO BE DETERMINED. PRELIMINARY INDUCTION STUDIES WILL BE EXTENDED TO QUANTITATE INDUCER UPTAKE OVER LONG AND SHORT INDUCTION PERIODS. INDUCTION OF THE CHIMERIC GENES WILL OCCUR THROUGH THE EXPOSURE OF AN EXOGENOUS INDUCER INTRODUCED THROUGH THE DRINKING WATER OR BY INTRAPERITONEAL INJECTION.