Analysis of DNA strand break repair using human knockout cells

我们最近开发了一种使用人类细胞系Nalm-6的系统，该系统能够快速生产人类基因敲除细胞。在这项研究中，我们使用该系统对与DNA链断裂修复有关的人类基因的功能进行了遗传分析。具体来说，我们破坏了一系列涉及Rad54、fanb、Mus81、Tdp1、Ku70/80和Lig3的基因，这些基因都在人类细胞中DNA单链和/或双链断裂修复中发挥一定作用。此外，我们试图制造尽可能多的双敲除突变细胞，特别是那些具有Lig4突变的细胞。利用这些突变体，我们成功地分析了细胞对包括顺铂和拓扑异构酶抑制剂在内的抗癌药物的敏感性，以及p53和BLM之间、Mus81和fanb之间的遗传相互作用，我们还对可能参与随机整合和/或基因靶向的DNA连接酶的sirna进行了基因敲低实验。结果表明，抑制非同源末端连接反应可以提高基因靶向的效率。我们的研究结果还表明，一定存在另一种不依赖于非同源末端连接途径的随机整合机制。

We recently developed a system using the human cell line Nalm-6 that enables rapid production of human gene knockout cells. In this study, we used this system to genetically analyze the function of human genes that have been implicated in DNA strand break repair. Specifically, we disrupted a series of genes involving those for Rad54, FancB, Mus81, Tdp1, Ku70/80, and Lig3, which all have some roles in DNA single and/or double-strand break repair in human cells. In addition, we tried to produce as many double-knockout mutant cells as possible, particularly those with a Lig4 mutation. Using these mutants, we have successfully analyzed the cellular sensitivity to anticancer agents involving cisplatin and topoisomerase inhibitors as well as the genetic interaction between p53 and BLM and between Mus81 and FancB, We also performed gene knockdown experiments with siRNAs for DNA ligases that are potentially involved in random integration and/or gene targeting. The results suggested that suppressing nonhomologous end-joining reactions could enhance the efficiency of gene targeting. Our findings also indicated that there must be another mechanism for random integration that does not rely on the nonhomologous end-joining pathway.