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.

已经开发出可诱导的CRE重组酶系统来绕过初始致命表型并提供后来的胚胎或成人表型。在这里，我们描述了一种转基因重组小鼠的产生，该小鼠通过靶向无处不在表达的ROSA26基因座，将四环素依赖性开关与广义的CRE重组酶表达结合在一起。使用通用和简化的基因递送系统开发了这种转基因菌株（R26RTTA-TRECRE），旨在通过整合这两个元素，反向四环素控制的反式激活器（RTTA）和RTTA诱导启动子在单个载体中促进有条件动物的产生。在这种转基因菌株中，内源性ROSA26启动子通过剪接受体位点驱动RTTA表达。四环素诱导启动子或四环素响应元件（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元素分离，驱动CRE重物组合酶表达。将这些小鼠与CRE报道菌株越过，在该菌株中，通过消除LOXP侧翼DNA序列激活了报告基因，表明在用TetracyCline -Cycline Abalog类似物（DoxyCycline）处理后，在各种产前发育范围内，在各种产前发育窗口中，CRE DNA介导的重组在各种产前发育窗口中被无处不在。在没有诱导剂的情况下，在某些组织中观察到背景CRE重组酶表达水平，主要是在胚胎晚期发育阶段。背景重组水平在发育过程中较低，在神经组织中最突出。在成年动物中无法有效诱导CRE重组酶表达。尽管成人组织中的RTTA mRNA水平较高，但强力霉素治疗后CRE重组酶mRNA水平仍然很低。因此，此处描述的小鼠应变提供了一种有价值的工具，可以通过在胚胎发育的定义阶段有效地失活其功能，从而进一步分析基因的功能。