Seidi Azadeh, Mie Masayasu, Kobatake Eiry
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
Biotechnol Lett. 2007 Sep;29(9):1315-22. doi: 10.1007/s10529-007-9406-6. Epub 2007 May 25.
A major limitation for the use of Cre recombinase is its toxicity and a lack of temporal control over its activity. We have developed a new recombination system using Cre recombinase alpha-complementation. Cre recombinase was divided and one fragment (beta) was introduced into cells between two loxP sites with a CMV promoter in the upstream. The gene of interest (EGFP) was positioned just downstream of this construct. Cre recombinase activity was recovered by adding the other part of the molecule (alpha) to cells as a protein fragment, as evidenced by the expression of EGFP under the control of the CMV promoter. The activity of fragmented cre reached 68% of that of the wild type enzyme at 1 microM alpha-protein.
使用Cre重组酶的一个主要限制是其毒性以及对其活性缺乏时间控制。我们开发了一种利用Cre重组酶α互补的新重组系统。将Cre重组酶进行分割，其中一个片段（β）被引入到两个loxP位点之间的细胞中，上游带有CMV启动子。感兴趣的基因（EGFP）位于该构建体的下游。通过将分子的另一部分（α）作为蛋白质片段添加到细胞中，可恢复Cre重组酶的活性，这由CMV启动子控制下EGFP的表达得以证明。在1 microM的α蛋白浓度下，片段化Cre的活性达到野生型酶活性的68%。
