Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells
Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells
复制标题
DOI：
10.1007/s12033-008-9098-8
复制
发表时间：
2009-02-01
影响因子：
2.6
通讯作者：
Murray, James D.
中科院分区：
文献类型：
--
作者：
Bertolini, Luciana R.;Bertolini, Marcelo;Murray, James D.
在哺乳动物体细胞中，通过同源DNA序列或基因打靶将外源DNA插入到特定的基因组位置是一个低效的过程。鉴于非同源末端连接(NHEJ)通路在哺乳动物细胞DNA双链断裂(DSB)修复中的关键作用，我们研究了NHEJ蛋白水平降低对基因靶向的影响。在这里，我们证明了RNAi在人HCT116细胞中瞬时击倒完整的NHEJ蛋白Ku70和Xrcc4对基因靶向/随机插入比率有显著影响。在RNAi治疗后，及时转染基于HPRT的靶向载体，导致随机整合事件减少70%，针对HPRT基因座的基因靶向增加33倍。这些发现支持了NHEJ蛋白在体内外源DNA整合中的作用，并证明了RNAi瞬时耗尽NHEJ蛋白是增加基因靶向事件频率的可行方法。了解外来DNA是如何整合到细胞基因组中的，对于推进生物技术和基因医学的战略非常重要。
The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell's genome is important to advance strategies for biotechnology and genetic medicine.