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

• 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
• 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' 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

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.

已经开发了诱导型Cre重组酶系统以绕过初始致死表型并提供进入后期胚胎或成体表型的途径。在这里，我们描述了通过靶向普遍表达的ROSA 26基因座将四环素依赖性开关与广义Cre重组酶表达相结合的转基因重组小鼠的产生。该转基因菌株（R26 rtTA-TRECre）是使用通用和简化的基因递送系统开发的，该系统旨在通过将两种元件（反向四环素控制的反式激活因子（rtTA）和rtTA诱导型启动子）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ROSA 26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应答元件（TRE）以与ROSA 26基因座相反的方向克隆并且通过5 kb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报告菌株杂交，其中报告基因通过消除loxP侧翼DNA序列而被激活，表明Cre DNA介导的重组在用四环素类似物（多西环素）处理后的各种产前发育窗口期间被普遍且有效地诱导。背景Cre重组酶的表达水平在一些组织中观察到的诱导剂的情况下，主要是在胚胎发育后期阶段。背景重组水平在发育过程中很低，在神经组织中最突出。Cre重组酶在成年动物中不能被有效地诱导表达。虽然rtTA mRNA水平在成体组织中很高，但多西环素处理后Cre重组酶mRNA水平仍然很低。因此，本文描述的小鼠品系提供了一种有价值的工具，通过允许在胚胎发育的整个限定阶段有效失活基因的功能，来进一步分析特定发育窗口期间基因的功能。