Inducible Cre expression through the Rosa 26 locus of recombinant mice

通过重组小鼠的 Rosa 26 位点诱导 Cre 表达

基本信息

批准号：
7733844
负责人：
Barry J Hoffer
金额：
$ 64.58万
依托单位：
NATIONAL INSTITUTE ON DRUG ABUSE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7733844
关键词：
3&apos Splice Site Adult Animal Model Animals Be++ element Beryllium Breeding Bypass Cells DNA DNA Sequence Development Doxycycline Elements Embryo Embryonic Development Gene Expression Gene Expression Regulation Generations Genes Genetic Genetic Recombination Human Individual Introns Link Mediating Messenger RNA Methods Modification Mouse Strains Mus Mutation Nerve Tissue Phenotype RNA Splicing Recombinants Regulation Reporter Reporter Genes Reporting Response Elements Rosa Silent Mutation Site Somatic Mutation Staging System TP53 gene Tetanus Helper Peptide Tetracycline Tetracycline Control Tetracyclines Time Tissues Trans-Activators Transcription Coactivator Transgenes Transgenic Animals Transgenic Model Transgenic Organisms analog design gene delivery system gene function improved in vivo interest knockout animal mature animal novel prenatal promoter recombinase segregation tool vector

项目摘要

Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. Here we described the generation of a transgenic recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain (R26rtTA-TRECre) was developed using a universal and simplified gene delivery system designed to facilitate the generation of conditional animals by integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter in a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter or tetracycline response element (TRE), cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain, in which the reporter gene is activated by the elimination of a loxP flanked DNA sequence, showed that Cre DNA mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows upon treatment with a tetracycline analog (doxycycline). Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages. Background recombination levels were low during development and most prominent in nervous tissue. Cre recombinase expression could not be effectively induced in adult animals. While rtTA mRNA levels were high in adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. Therefore, the mouse strain described here provides a valuable tool to further analyze the function of genes during specific developmental windows, by allowing the effective inactivation of their function throughout defined stages of embryonic development.

诱导型 Cre 重组酶系统已被开发用于绕过最初的致死表型并提供获得后来的胚胎或成体表型的机会。在这里，我们描述了转基因重组小鼠的产生，该小鼠通过靶向普遍表达的 ROSA26 基因座，将四环素依赖性开关与广义 Cre 重组酶表达相结合。该转基因菌株 (R26rtTA-TRECre) 是使用通用且简化的基因传递系统开发的，该系统旨在通过将反向四环素控制的反式激活子 (rtTA) 和 rtTA 诱导型启动子这两个元件整合到单个载体中来促进条件动物的产生。在该转基因菌株中，内源性 ROSA26 启动子通过剪接受体位点驱动 rtTA 表达。四环素诱导型启动子或四环素反应元件 (TRE)，以与 ROSA26 基因座相反的方向克隆，并通过 5kb 人 p53 内含子与 rtTA 元件分开，驱动 Cre 重组酶表达。将这些小鼠与 Cre 报告菌株（其中报告基因通过消除 loxP 侧翼 DNA 序列而激活）进行杂交，结果表明，在用四环素类似物（强力霉素）治疗后，在各个产前发育窗口期间，Cre DNA 介导的重组被普遍有效地诱导。）。背景在没有诱导物的情况下，在一些组织中观察到 Cre 重组酶表达水平，主要是在胚胎发育后期。背景重组水平在发育过程中较低，并且在神经组织中最为突出。在成年动物中不能有效诱导Cre重组酶表达。虽然成人组织中 rtTA mRNA 水平较高，但多西环素治疗后 Cre 重组酶 mRNA 水平仍然较低。因此，这里描述的小鼠品系提供了一个有价值的工具，通过允许在胚胎发育的整个确定阶段有效失活基因的功能，进一步分析特定发育窗口期间基因的功能。