Inducible Cre expression through the Rosa 26 locus of recombinant mice

已开发出可诱导的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人表型的途径。在这里，我们描述了通过靶向无处不在表达的ROSA26位点，将四环素依赖开关与广义Cre重组酶表达结合起来的转基因重组小鼠的产生。该转基因菌株（R26rtTA-TRECre）是利用通用和简化的基因传递系统开发的，旨在通过将逆转录四环素控制的反式激活子（rtTA）和rtTA诱导启动子这两种元素整合在一个载体上，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。四环素诱导启动子或四环素反应元件（TRE），克隆于ROSA26位点的相反方向，通过5kb的人p53内含子与rtTA元件分离，驱动Cre重组酶的表达。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DNA序列而激活，结果表明，在四环素类似物（强力霉素）处理的不同产前发育窗口期间，Cre DNA介导的重组普遍有效地诱导。在没有诱导剂的情况下，在一些组织中观察到Cre重组酶的表达水平，主要是在胚胎发育后期。背景重组水平在发育过程中较低，在神经组织中最为突出。Cre重组酶在成年动物体内不能有效诱导表达。虽然rtTA mRNA水平在成人组织中很高，但在多西环素治疗后，Cre重组酶mRNA水平仍然很低。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特定阶段有效地失活它们的功能，进一步分析基因在特定发育窗口中的功能。

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.