Toxicity of ligand-dependent Cre recombinases and generation of a conditional Cre deleter mouse allowing mosaic recombination in peripheral tissues
Toxicity of ligand-dependent Cre recombinases and generation of a conditional Cre deleter mouse allowing mosaic recombination in peripheral tissues
复制标题
DOI：
10.1152/physiolgenomics.00019.2007
复制
发表时间：
2007-09-19
影响因子：
4.6
通讯作者：
Bockamp, Ernesto
中科院分区：
文献类型：
--
作者：
Hameyer, Dorothe;Loonstra, Ate;Bockamp, Ernesto
配体激活的Cre重组酶被广泛应用于体外和条件性小鼠模型的基因功能研究。为了比较配体依赖的Cre重组酶，将不同的Cre雌激素受体融合基因导入胚胎干细胞(ES细胞)的rosa26基因座，并检测其遗传毒性和重组效率。在所测试的重组酶中，CreERT2变异体没有毒性，并且对配体诱导高度敏感。为了在小鼠中结构性地表达CreERT2，也为了阐明CreERT2系统是否显示背景活性，我们在rosa26基因座的控制下产生了一个含有CreERT2编码区的敲入小鼠系。对这只具有不同报告品系的rosa26-CreERT2缺失小鼠的分析表明，在胚胎中普遍存在重组，在外周和造血组织中也有部分重组，但在大脑中没有有效的CreERT2表达。此外，使用流式细胞术，我们发现在未诱导的双反式基因rosa26-CreERT2/EGFP报告小鼠中存在低水平的背景重组。为了确定背景活动是否对使用ROSA26-CreERT2缺失器进行条件性体内实验构成一般问题，我们使用了敏感的条件性皮肤癌模型。在本实验中，癌症诱导完全限于诱导的双基因CreERT2/K-RAS(V12)小鼠，而未诱导的对照组动物没有表现出任何癌症迹象，表明ROSA-CreERT2系统在体内调节条件性基因表达方面是有用的。Rosa26-CreERT2缺失菌株将是一种方便的实验工具，用于研究需要部分诱导外周组织重组的情况下的基因功能，并将有助于揭示以前未知或未知的表型。
Ligand-activated Cre recombinases are widely used for studying gene function in vitro and in conditional mouse models. To compare ligand-dependent Cre recombinases, different Cre estrogen receptor fusions were introduced into the ROSA26 locus of embryonic stem (ES) cells and assayed for genotoxicity and recombination efficiency. Of the tested recombinases, the CreERT2 variant showed no toxicity and was highly responsive to ligand induction. To constitutively express CreERT2 in mice and also to clarify whether the CreERT2 system displays background activity, we generated a knock-in mouse line harboring the CreERT2 coding region under the control of the ROSA26 locus. Analysis of this ROSA26-CreERT2 deleter mouse with different reporter strains revealed ubiquitous recombination in the embryo and partial recombination in peripheral and hematopoietic tissues but no effective CreERT2 expression in the brain. Furthermore, using flow cytometry, we found low-level background recombination in noninduced bitransgenic ROSA26-CreERT2/ EGFP reporter mice. To determine whether background activity poses a general problem for conducting conditional in vivo experiments with the ROSA26-CreERT2 deleter, we used a sensitive conditional skin cancer model. In this assay, cancer induction was completely restricted to induced bitransgenic CreERT2/K-Ras(V12) mice, whereas noninduced control animals did not show any sign of cancer, indicating the usefulness of the ROSA-CreERT2 system for regulating conditional gene expression in vivo. The ROSA26-CreERT2 deleter strain will be a convenient experimental tool for studying gene function under circumstances requiring partial induction of recombination in peripheral tissues and will be useful for uncovering previously unknown or unsuspected phenotypes.