Sauer B, Henderson N
E. I. du Pont de Nemours and Company, Inc., Central Research and Development Department, Wilmington, DE 19898.
Proc Natl Acad Sci U S A. 1988 Jul;85(14):5166-70. doi: 10.1073/pnas.85.14.5166.
The Cre protein encoded by the coliphage P1 is a 38-kDa protein that efficiently promotes both intra- and intermolecular synapsis and recombination of DNA both in Escherichia coli and in vitro. Recombination occurs at a specific site, called lox, and does not require any other protein factors. The Cre protein is shown here also to be able to cause synapsis of DNA and site-specific recombination in a mammalian cell line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallothionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. One such substrate is a circular plasmid with two directly repeated lox sites (lox2) flanking a marker gene and was introduced into cells by Ca3(PO4)2 transformation. As a second substrate we used a pseudorabies virus (a herpesvirus) containing a lox2 insertion designed to provide a sensitive detection system for recombination. In both cases, site-specific recombination in vivo is dependent on the presence of the Cre protein and occurs specifically at the 34-base-pair lox sites. These results demonstrate the controlled site-specific synapsis of DNA and recombination by a prokaryotic protein in mammalian cells and suggest that Cre-mediated site-specific recombination may be a useful tool for understanding and modulating genome rearrangements in eukaryotes.
由大肠杆菌噬菌体P1编码的Cre蛋白是一种38 kDa的蛋白质，它在大肠杆菌体内和体外均能有效地促进DNA的分子内和分子间联会及重组。重组发生在一个特定的位点，称为lox，且不需要任何其他蛋白质因子。本文还表明，Cre蛋白能够在哺乳动物细胞系中引发DNA联会和位点特异性重组。构建了一个稳定的小鼠细胞系，该细胞系在Cd2+诱导型金属硫蛋白I基因启动子的控制下表达Cre蛋白。使用含有两个同向重复lox位点的DNA底物监测DNA重组。其中一种底物是一个环状质粒，在一个标记基因两侧有两个同向重复的lox位点（lox2），通过Ca3(PO4)2转化法导入细胞。作为第二种底物，我们使用了一种含有lox2插入片段的伪狂犬病病毒（一种疱疹病毒），旨在为重组提供一个灵敏的检测系统。在这两种情况下，体内的位点特异性重组都依赖于Cre蛋白的存在，并且特异性地发生在34个碱基对的lox位点。这些结果证明了原核蛋白在哺乳动物细胞中对DNA进行可控的位点特异性联会和重组，表明Cre介导的位点特异性重组可能是理解和调控真核生物基因组重排的有用工具。
