DNA breakage associated with targeted gene alteration directed by DNA oligonucleotides
DNA breakage associated with targeted gene alteration directed by DNA oligonucleotides
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DOI：
10.1016/j.mrfmmm.2009.05.004
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发表时间：
2009-10-02
影响因子：
2.3
通讯作者：
Kmiec, Eric B.
中科院分区：
文献类型：
--
作者：
Bonner, Melissa;Kmiec, Eric B.
了解单链寡核苷酸（ODN）引发靶向核苷酸交换（TNE）的机制对于实现最佳纠正效率和医疗适用性至关重要。先前已经表明，将ODN引入细胞会导致DNA损伤反应途径的激活，但尚未对DNA水平上产生的损伤进行评估。DNA断裂的标志蛋白H2AX的激活表明，在靶向基因改变（TGA）反应中可能发生双链断裂（DSB），我们使用具有单个整合突变eGFP基因的人HCT116细胞系作为模型系统。我们证明了DNA链断裂发生在特定的ODN。单链和双链DNA的切割取决于加入到反应中的ODN的水平，本文讨论了ODN依赖的DSB形成的可能机制，作为TGA的功能(C) 2009 Elsevier B.V.
Understanding the mechanism by which single-stranded oligonucleotides (ODNs) elicit targeted nucleotide exchange (TNE) is imperative to achieving optimal correction efficiencies and medical applicability It has been previously shown that introduction of an ODN into cells results in the activation of DNA damage response pathways, but there has been no evaluation of the damage created at the level of the DNA. The activation of H2AX, a hallmark protein of DNA breakage, suggests that a double-strand break (DSB) could be occurring during the targeted gene alteration (TGA) reaction Using the human HCT116 cell line with a single integrated mutant eGFP gene as our model system. we demonstrate that the DNA strand breakage occurs when a specific ODN. designed to direct TGA, is transfected into the cells Both single- and double-stranded DNA cleavage is observed dependent on the level of ODN added to the reaction Possible mechanisms of ODN-dependent DSB formation, as a function of TGA, are discussed herein (C) 2009 Elsevier B.V. All rights reserved