Brocard J, Warot X, Wendling O, Messaddeq N, Vonesch J L, Chambon P, Metzger D
Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collège de France, Strasbourg, France.
Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14559-63. doi: 10.1073/pnas.94.26.14559.
The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type will facilitate studies of gene function and the generation of animal models for human diseases. We have shown previously that conditional recombination-excision between two loxP sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ERT), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ERT under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level ( approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ERT in a given cell type, we have now crossed Cre-ERT-expressing mice with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ERT. These results indicate that cell-specific expression of Cre-ERT in transgenic mice can be used for efficient tamoxifen-dependent, Cre-mediated recombination at loci containing loxP sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.
在特定时间、特定细胞类型中高效引入特定基因的体细胞突变，将有助于基因功能的研究以及人类疾病动物模型的建立。我们之前已经表明，通过使用与人雌激素受体的突变配体结合域融合的Cre重组酶（Cre-ERT），可以在小鼠中实现两个loxP位点之间的条件性重组切除，该重组酶可结合他莫昔芬但不结合雌激素。向在巨细胞病毒启动子控制下表达Cre-ERT的转基因小鼠给予他莫昔芬后，在多个组织中诱导了DNA切除。然而，切除效率在不同组织间有所差异，在皮肤中获得了最高水平（约40%）。为了确定Cre-ERT在特定细胞类型中介导的切除效率，我们现在已将表达Cre-ERT的小鼠与报告基因小鼠杂交，在报告基因小鼠中，大肠杆菌β-半乳糖苷酶的表达可通过Cre介导的重组来诱导。在6至8周龄双转基因小鼠的表皮细胞水平上分析了这种重组的效率和动力学。我们表明，在给予他莫昔芬后的几天内，基本上所有表达Cre-ERT的表皮细胞中都发生了位点特异性切除。这些结果表明，转基因小鼠中Cre-ERT的细胞特异性表达可用于在含有loxP位点的基因座上进行高效的他莫昔芬依赖性、Cre介导的重组，从而以时空控制的方式产生位点特异性体细胞突变。
