Spatial and temporal control of the mouse genome by lac

描述（由申请人提供）：本提案的总体目标是 小鼠基因表达的全基因组时空调控 缺乏操纵子-阻遏子系统。我们改进后的系统已经被用于 调节鼠转基因。为了验证lac操纵子-阻遏子系统作为 有效的替代现有的条件诱变策略，我们 我建议产生携带lac操纵基因元件靶向插入的小鼠 在内源性Arc和Hdh基因内（aim 1）。为了实现这一目标，我们将 开发一种新的插入载体，设计用于捕获包含lac的内含子， 操纵子控制区（lac OCRR）和绿色荧光蛋白（GFP）标签。 为了在ES细胞的全基因组基因陷阱中使用该载体之前对其进行测试， 我们将用lac OCR-GFP插入靶向Hdh基因的第一个内含子 载体以产生lac操纵子（lacO）修饰的Hdh等位基因。我们还将 通过将ac操纵基因元件靶向至 ARC启动子。我们的目标是利用在修改基因中获得的经验 抑制两种主要的启动子类别（富含G/C和缺乏TATA盒; Hdh和 含TATA的; Arc）以获得对任何内源性基因的表达的控制。 通过有针对性地或随机地插入LAC操作符。基因空间控制 表达将由乳糖阻遏物（laclR）的模式赋予 表情一个基因捕获的ES细胞克隆库， 用GFP标记lacIR的整合将提供一组试剂 可以用来构建具有不同模式的抑制小鼠品系， 目的2.用这些ES细胞克隆产生的小鼠将 补充现有的转基因，并提出了LaclR的靶向插入 编码序列转化为β-肌动蛋白（通用表达），鱼精蛋白 （睾丸特异性表达）和Camk 2a（成人前脑特异性表达） 基因.与IPTG组合，这些ES克隆和小鼠系将提供 这些试剂能够精确控制小鼠基因在何时何地 是表达。此外，使用lac系统来调节Hdh和Arc将 这使我们能够探索这些基因在人类发育过程中发挥重要作用的假设。 突触发生和突触可塑性和精子发生在成年人（目的 第三章

DESCRIPTION (provided by applicant): 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)