Subtle gene modification in mouse ES cells: evidence for incorporation of unmodified oligonucleotides without induction of DNA damage.
Subtle gene modification in mouse ES cells: evidence for incorporation of unmodified oligonucleotides without induction of DNA damage.
复制标题
DOI：
10.1093/nar/gkq589
复制
发表时间：
2010-11
影响因子：
14.9
通讯作者：
te Riele H
中科院分区：
文献类型：
--
作者：
Aarts M;te Riele H
单链寡脱氧核苷酸（ssODNs）基因打靶技术是一种很有前途的小鼠胚胎干细胞（ESCs）定点基因修饰技术。我们已经开发了一种携带突变的EGFP报告基因的ESC系，以监测暴露于ssODNs后不久的基因校正事件。我们使用该系统来比较由正义或反义ssODNs校正的细胞的外观和命运。与反义ssODN相比，具有正义ssODN的绿色荧光细胞的较慢出现与ssODN物理掺入基因组一致。因此，先前由其他人报道的反义ssODNs的优势可能是EGFP报告子的早期读出的假象。重要的是，通过未修饰的ssODNs的基因校正仅轻度影响靶细胞的活力，并且不诱导基因组DNA双链断裂（DSB）。相比之下，由于基因组DSB的诱导，通过硫代磷酸酯（PTO）键末端保护的ssODN在校正的和未校正的细胞中引起H2AX磷酸化增加和细胞周期进展受损。我们的研究结果表明，使用未经修饰的而不是PTO末端保护的ssODN允许稳定的基因修饰，而不损害细胞的基因组完整性，这对于ssODN介导的基因靶向（胚胎）干细胞的应用至关重要。
Gene targeting by single-stranded oligodeoxyribonucleotides (ssODNs) is a promising tool for site-specific gene modification in mouse embryonic stem cells (ESCs). We have developed an ESC line carrying a mutant EGFP reporter gene to monitor gene correction events shortly after exposure to ssODNs. We used this system to compare the appearance and fate of cells corrected by sense or anti-sense ssODNs. The slower appearance of green fluorescent cells with sense ssODNs as compared to anti-sense ssODNs is consistent with physical incorporation of the ssODN into the genome. Thus, the supremacy of anti-sense ssODNs, previously reported by others, may be an artefact of early readout of the EGFP reporter. Importantly, gene correction by unmodified ssODNs only mildly affected the viability of targeted cells and did not induce genomic DNA double-stranded breaks (DSBs). In contrast, ssODNs that were end-protected by phosphorothioate (PTO) linkages caused increased H2AX phosphorylation and impaired cell cycle progression in both corrected and non-corrected cells due to induction of genomic DSBs. Our results demonstrate that the use of unmodified rather than PTO end-protected ssODNs allows stable gene modification without compromising the genomic integrity of the cell, which is crucial for application of ssODN-mediated gene targeting in (embryonic) stem cells.
登录
查看更多内容
影响因子：
14.8
作者：
Olive, Peggy L.;Banath, Judit P.
通讯作者：
Banath, Judit P.
影响因子：
9.2
作者：
Paull, TT;Rogakou, EP;Bonner, WM
通讯作者：
Bonner, WM
影响因子：
3.5
作者：
Papaioannou, Ioannis;Disterer, Petra;Owen, James S.
通讯作者：
Owen, James S.
DOI：
10.1016/j.mrfmmm.2009.05.004
发表时间：
2009-10-02
影响因子：
2.3
作者：
Bonner, Melissa;Kmiec, Eric B.
通讯作者：
Kmiec, Eric B.
影响因子：
--
作者：
Igoucheva, Olga;Alexeev, Vitali;Rubin, Emanuel
通讯作者：
Rubin, Emanuel