The overall goal of this proposal is the genome-wide spatial and temporal regulation of murine gene expression by the lac operator-repressor system. Our modified system is already being used to regulate murine transgenes. To validate the lac operator-repressor system as an efficient alternative to existing conditional mutagenesis strategies, we propose generating mice carrying targeted insertions of lac operator elements within the endogenous Arc and Hdh genes (aim 1). To accomplish this, we will develop a novel insertion vector designed to trap introns that includes a lac operator control region (lac OCRR) and a green fluorescent protein (GFP) tag. To test this vector before employing it in a genome-wide gene trap in ES cells, we will target the first intron of the Hdh gene with the lac OCR-GFP insertion vector to generate a lac operator (lacO) modified Hdh allele. We will also generate a LacO-modified allele of Arc by targeting ac operator elements into the ARC promoter. Our goal is to use the experience gained in modifying genes repressing two major promoter classes (G/C-rich and lacking TATA box; Hdh and TATA-containing; Arc) to gain control over the expression of any endogenous ene by targeted or random insertion of lac operators. Spatial control of gene expression will be conferred by the pattern of lac repressor (laclR) expression. A library of gene-trapped ES cell clones harboring random integration of laclR that are tagged with GFP will provide a set of reagents that can be used to construct lines of repressor mice with different patterns of LaclR expression (Aim 2). Mice generated with these ES cell clones will complement existing transgenic, and proposed targeted insertions of the LaclR coding sequences into the beta-actin (universal expression), protamine (testis-specific expression), and Camk2a (adult forebrain-specific expression) genes. In combination with IPTG, these ES clones and mouse lines will provide the reagents to achieve precise control over both when and where a murine gene is expressed. Moreover, use of the lac system to regulate Hdh and Arc will allow us to explore the hypothesis that these genes play essential roles during synaptogenesis and in synaptic plasticity and spermatogenesis in the adult (aim 3)
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