Spatial and temporal control of the mouse genome by lac

lac对小鼠基因组的时空控制

• 批准号: 6447159
• 负责人: HEIDI J. SCRABLE
• 金额: $ 13.71万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6447159
• 关键词: cell death; cell differentiation; cell population study; developmental genetics; gene expression; gene targeting; genetic regulation; genetically modified animals; green fluorescent proteins; immunocytochemistry; immunofluorescence technique; laboratory mouse; lac operon; neural degeneration; neural plasticity; neurogenesis; regulatory gene; spermatogenesis; stem cells; synaptogenesis; tissue resource /registry; transfection /expression vector; western blottings

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)

描述（由申请人提供）：该提案的总体目标是 小鼠基因表达的全基因组空间和时间调控 lac 操纵子阻遏系统。我们修改后的系统已被用于 调节小鼠转基因。验证 lac 操纵子抑制系统作为 现有条件诱变策略的有效替代方案，我们 建议生成携带 lac 操作元件定向插入的小鼠 内源性 Arc 和 Hdh 基因（目标 1）。为了实现这一目标，我们将 开发一种新颖的插入载体，旨在捕获包含 lac 的内含子 操作员控制区 (lac OCRR) 和绿色荧光蛋白 (GFP) 标签。 为了在将其用于 ES 细胞的全基因组基因陷阱之前测试该载体， 我们将通过 lac OCR-GFP 插入来定位 Hdh 基因的第一个内含子 向量生成 lac 算子 (lacO) 修饰的 Hdh 等位基因。我们还将 通过将 ac 操作符元素定位到 Arc 生成 LacO 修饰的等位基因 ARC 启动子。我们的目标是利用在修改基因中获得的经验 抑制两个主要启动子类别（富含 G/C 和缺乏 TATA 盒；Hdh 和 含TATA； Arc）来控制任何内源烯的表达 通过有针对性或随机插入 lac 运算符。基因的空间控制 表达将由 lac 阻遏物 (laclR) 的模式赋予 表达。基因捕获的 ES 细胞克隆文库，包含随机 整合带有 GFP 标记的 laclR 将提供一组试剂 可用于构建具有不同模式的阻遏小鼠品系 LaclR 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，并提出 LaclR 的定向插入 β-肌动蛋白（通用表达）、鱼精蛋白的编码序列 （睾丸特异性表达）和 Camk2a（成人前脑特异性表达） 基因。与 IPTG 结合，这些 ES 克隆和小鼠品系将提供 用于精确控制鼠基因何时何地出现的试剂 被表达。此外，使用 lac 系统来调节 Hdh 和 Arc 将 让我们探索以下假设：这些基因在 成人的突触发生以及突触可塑性和精子发生（目标 3）