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Show Faculty Sponsor Mario Capecchi GENERATION OF MOUSE EMBRYONIC STEM CELLS FOR TIME-AND TISSUE-SPECIFIC CRE TRANSGENE EXPRESSION IN LIVING MICE Mark S. Hansen, Charles Keller, (Mario R. Capecchi) Department of Genetics, University of Utah Molecular techniques allow for genetic modifications of the mouse, in vivo. To control the expression or ablation of any gene, an ectopic site specific Cre recombinase with tissue specificity and temporal specificity is used. Unfortunately, Cre is often "turned on" too early, creating undesired background effects. Creating a stringent system in which Cre expression is temporally inducible will be extremely helpful in controlling gene expression at differ-ent developmental stages. Gene targeting was used to modify the 3' untranslated region of the neuromuscular-restricted Pax7 gene. Two targeting vectors were designed. For the first tetra- cycline-inducible vector, we inserted a complex cassette down- stream of the Pax7 stop codon, but upstream of the Pax7 polyad- enylation signal. This cassette consisted of an internal ribosome entry site, a tetracycline transactivator, a tetracycline suppressor, and a tetracycline response element driving Cre expression. The second tamoxifen-inducible vector utilized a less complex cas-sette containing an internal ribosome entry site and a Cre-estro-gen receptor chimeric fusion gene. Following electroporation of these targeting vectors into mouse embryonic stems cells under positive and negative selection, we screened candidate embry-onic stem cell clones by Southern hybridization. Clones with the correct integration were obtained and potential germline founder mice are being screened. |
