Double-stranded break can be repaired by single-stranded oligonucleotides via the ATM/ATR pathway in mammalian cells
Double-stranded break can be repaired by single-stranded oligonucleotides via the ATM/ATR pathway in mammalian cells
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DOI：
10.1089/oli.2007.0093
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发表时间：
2008-03-01
期刊：
影响因子：
--
通讯作者：
Huang, Jian-Dong
中科院分区：
文献类型：
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作者：
Wang, Zai;Zhou, Zhong-Jun;Huang, Jian-Dong
单链寡核苷酸（SSO）介导的基因修饰是一种新开发的工具，用于哺乳动物细胞中的位点特异性基因修复；然而，校正后的细胞总是显示 G2/M 停滞并且不能分裂形成集落。这种现象和尚不清楚的机制严重挑战了该技术的未来应用。在这项研究中，我们开发了一种基于双链断裂 (DSB) 诱导的高效 SSO 介导的 DNA 修复系统。我们生成了一个突变型 EGFP 基因，插入长度为 24 bp 至 1.6 kb，作为整合到哺乳动物细胞系中的报告基因。在插入附近位点进行 DSB 诱导后，SSO 被成功用于删除插入片段。我们证明这个过程依赖于 ATM/ATR 途径。重要的是，修复后的细胞克隆是可行的。还研究了缺失长度、SSO 长度、链偏向和 SSO 修饰对基因修复频率的影响。
Single-stranded oligonucleotide (SSO)-mediated gene modification is a newly developed tool for site-specific gene repair in mammalian cells; however, the corrected cells always show G2/M arrest and cannot divide to form colonies. This phenomenon and the unclear mechanism seriously challenge the future application of this technique. In this study, we developed an efficient SSO-mediated DNA repair system based on double-stranded break (DSB) induction. We generated a mutant EGFP gene with insertions of 24 bp to 1.6 kb in length as a reporter integrated in mammalian cell lines. SSOs were successfully used to delete the insertion fragments upon DSB induction at a site near the insertion. We demonstrated that this process is dependent on the ATM/ATR pathway. Importantly, repaired cell clones were viable. Effects of deletion length, SSO length, strand bias, and SSO modification on gene repair frequency were also investigated.