Generalized tetracycline induced Cre recombinase expression through the ROSA26 locus of recombinant mice
Generalized tetracycline induced Cre recombinase expression through the ROSA26 locus of recombinant mice
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DOI：
10.1016/j.jneumeth.2008.08.024
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发表时间：
2009-01-15
影响因子：
3
通讯作者：
Tomac, Andreas C.
中科院分区：
文献类型：
--
作者：
Backman, Cristina M.;Zhang, YaJun;Tomac, Andreas C.
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重组小鼠的产生。该转基因菌株是使用简化的基因递送系统开发的，该基因递送系统将反向四环素控制的反式激活因子（rtTA）和rtTA诱导型启动子这两种元件整合到单个载体中。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驱动Cre重组酶表达，该启动子以与ROSA 26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。将这些小鼠与Cre报告菌株杂交表明，Cre DNA介导的重组在各种产前发育窗口期间普遍有效地诱导。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和成年动物。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水平在发育和成人组织中很高，但多西环素治疗后Cre重组酶mRNA水平仍然很低。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定义阶段有效失活基因功能，进一步分析特定发育窗口期间基因的功能。由爱思唯尔公司出版
Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. Here we describe the generation of a recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain was developed using a simplified gene delivery system integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter into a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter, cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5 kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain showed that Cre DNA-mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows. Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages and in adult animals. 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 developmental and adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. 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. Published by Elsevier B.V.