Spatial and temporal control of the mouse genome by lac

描述(由申请人提供)：本建议书的总体目标是 小鼠基因表达的全基因组时空调控 Lac操纵者-抑制器系统。我们修改后的系统已经被用来 调控小鼠的转基因。要验证lac操纵子-抑制子系统是否为 现有条件突变策略的有效替代方案，我们 建议产生携带Lac操纵子元件的靶向插入的小鼠 在内源性Arc和HDH基因内(目标1)。为了实现这一目标，我们将 开发一种新型的插入载体，旨在捕获含有Lac的内含子 操作员控制区(Lac OCRR)和绿色荧光蛋白(GFP)标签。 为了在将该载体应用于ES细胞的全基因组基因陷阱之前对其进行测试， 我们将针对hdh基因的第一个内含子插入lac OCR-GFP 载体以产生Lac操作符(LACO)修饰的HDH等位基因。我们还将 通过靶向Ac操作符元件生成Arc的LACO修饰的等位基因 ARC的发起人。我们的目标是利用在修改基因方面获得的经验 抑制两个主要的启动子类(富含G/C和缺乏TATA盒；HDH和 包含TATA的；Arc)以获得对任何内源烯的表达的控制 通过定向或随机插入Lac操作符。基因的空间调控 表达将由lac抑制物(LaclR)的模式授予 表情。携带随机基因的ES细胞克隆文库 整合标记有GFP的laclR将提供一组试剂 可以用来构建具有不同模式的抑制子小鼠的系 LaclR的表达(目标2)。用这些ES细胞克隆产生的小鼠将 补充现有的转基因，并建议有针对性地插入LaclR 编码序列为β-肌动蛋白(通用表达)，鱼精蛋白 (睾丸特异表达)和Camk2a(成人前脑特异表达) 基因。与IPTG相结合，这些ES克隆和鼠系将提供 这些试剂可以在何时何地精确控制小鼠基因 被表达出来。此外，使用Lac系统来调节HDH和Arc将 允许我们探索这样一种假设，即这些基因在 成人的突触发生及突触可塑性和精子发生(目的 3)

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)