Analysis of the role of homology arms in gene-targeting vectors in human cells.
Analysis of the role of homology arms in gene-targeting vectors in human cells.
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DOI：
10.1371/journal.pone.0108236
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Adachi N
中科院分区：
文献类型：
--
作者：
Ishii A;Kurosawa A;Saito S;Adachi N
靶向载体随机整合到基因组中是人类体细胞基因靶向的主要障碍。非同源末端连接（NHEJ）是修复 DNA 双链断裂的主要途径，被认为是大多数随机整合事件的原因；然而，缺少关键的 NHEJ 连接酶 DNA 连接酶 IV (LIG4) 并不会显着降低人类细胞中靶向载体的随机整合频率，表明通过不依赖 LIG4 的机制发生稳健的整合事件。为了深入了解不依赖 LIG4 的随机整合的机制和稳健性，我们采用了各种类型的靶向载体来检查它们在 LIG4 熟练和缺乏的人类细胞系中的整合频率。我们发现靶向载体的整合频率与同源臂的长度以及臂中存在的重复DNA序列（尤其是SINE）的数量密切相关。这种相关性在 LIG4 缺陷细胞中很突出，但在 LIG4 丰富细胞中也可见，从而提供证据表明，即使 NHEJ 功能正常，LIG4 独立的随机整合也经常发生。我们的结果共同表明，传统靶向载体的随机整合频率很大程度上受到同源臂的影响，同源臂通常含有重复的DNA序列，无论是否存在功能性NHEJ，这些序列都有助于促进人类细胞中不依赖LIG4的随机整合。
Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4), the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.
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影响因子：
14.9
作者：
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通讯作者：
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DOI：
10.1038/nrg3117
发表时间：
2011-11-29
期刊：
Nature reviews. Genetics
影响因子：
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作者：
通讯作者：
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影响因子：
3.8
作者：
Banuelos, C. A.;Banath, J. P.;Olive, P. L.
通讯作者：
Olive, P. L.