Development of a highly efficient method to integrate genes into genome of higher eukaryotic cells by homologous recombination

本研究的目的是确定参与被BLM抑制的同源重组的蛋白质，并建立一种方法，使我们能够通过抑制BLM功能来高效地进行靶向整合。尽管我们没有成功地找到BLM的良好抑制剂，但我们获得了关于BLM的功能以及在没有BLM1的情况下发生的重组的重要信息。通过建立和分析双基因敲除的DT40细胞、BLM和RAD54、RAD51、ATM、Mre11、RECQL1或RECQL5，结果表明，在BLM破坏的细胞中，增加的姐妹染色单体交换(SCE)大部分是通过同源重组形成的，ATM和MRE 11对姐妹染色单体交换的贡献相对较小。相反，ATM极大地促进了靶向整合，这种整合的频率因BLM基因2的中断而增加。对DT 40细胞的分析也表明，RecQL5参与了BLM基因敲除细胞的靶向整合。对酵母SGS1(BLM的酵母同源物)基因干扰物的分析表明，SGS1在正常条件下具有抑制重组的功能，但在损伤诱导条件下具有诱导重组的功能，可能与拓扑异构酶III相互作用。

The aim of this study is to identify the proteins involved in the homologous recombination that is suppressed by BLM and to establish a method that enable us to perform targeted integration highly efficiently by inhibiting BLM function. Although we could not succeed to find good inhibitors for BLM, we obtained important information concerning the function of BLM and the recombination that occurs in the absence of BLM.1. By generating and analyzing double gene knockout DT40 cells, BLM and RAD54, RAD51, ATM, MRE11, RECQL1, or RECQL5, it was indicated that that the majority of increased sister chromatid exchanges (SCEs) in BLM disrupted cells are formed via homologous recombination, and the contribution of ATM and MRE 11 to the SCE is relatively small. In contrast, ATM greatly contributed to the targeted integration the frequency of which was increased by disruption of BLM gene.2. The analysis using DT 40 cells also indicated that RecQL5 was involved in the targeted integration in BLM gene knockout cells.3. An analysis of yeast SGS1 (the yeast homologue of BLM) gene disruptants indicated that Sgs1 functions to suppress recombination under normal conditions but functions to induce recombination under damage-inducing conditions probably interacting with topoisomerase III.