Homologous Recombination Assay

Abstract

Repair of double strand break by homologous recombination was examined using U2OS cells or RG37 cells harbouring specific substrate developed by Puget et al. (2005) and Dumay et al. (2006), respectively, to measure the repair of DNA double strand breaks by homologous recombination. The substrate is composed of two inactive copies of the GFP gene. The upstream copy is inactive due to the absence of promoter, the downstream copy present a promoter but is inactivated by the insertion of the sequence coding for the recognition site of the I-SceI enzyme. The substrate is stably expressed in cells after its insertion in the genome and present as a unique copy. The unique DNA double strand break is then induced by the expression of the I-SceI enzyme after cell transfection with a plasmid coding for the I-SceI enzyme., 使用由Puget等人开发的含有特异性底物的U2OS细胞或RG37细胞检查通过同源重组修复双链断裂。 （2005）和Dumay等人 （2006），分别通过同源重组来测量DNA双链断裂的修复。 底物由GFP基因的两个无活性拷贝组成。 上游拷贝由于不存在启动子而不活跃，下游拷贝存在启动子，但通过插入编码I-SceI酶的识别位点的序列而失活。 底物在插入基因组后稳定地表达在细胞中，作为唯一拷贝存在。 然后通过用编码I-SceI酶的质粒转染细胞后，通过I-SceI酶的表达来诱导独特的DNA双链断裂。