Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.
Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.
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DOI：
10.1371/journal.pone.0020514
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Weinstock DM
中科院分区：
文献类型：
--
作者：
Fung H;Weinstock DM
体外细胞培养条件的不同会极大地影响干细胞的生理学。我们试图建立一种方法来测量化学、环境和遗传操作对多能和体细胞中单个DNA双链断裂(DSB)修复精度的影响。哺乳动物细胞中的双链断裂主要通过同源重组(HR)或非同源末端连接(NHEJ)修复。在大多数情况下，以前对人类细胞中DSB修复的研究利用了非特异性的碎裂原，如电离辐射，这是高度非生理性的，或在随机整合的报告中进行检测修复。在随机积分后测量修复可能会受到修复效率和精确度的特定位点的影响。我们发现，基于DSB在基因组中的位置，单个DSB的HR频率在其他相同基因的人类胚胎干细胞(HESCs)之间差异高达20倍。为了克服位点特异性对DSB修复的影响，我们使用锌指核酸酶有效地将DSB修复报告靶向hESCs和一组体细胞系中的安全港基因座。我们证明，与体细胞人类细胞或小鼠胚胎干细胞相比，hESCs中靶向DSB的修复是高度精确的。携带靶向报告基因的人胚胎干细胞分化为星形胶质细胞会降低修复的效率和精度。因此，单个DSB的修复表型可以根据基因组内的损伤位置或细胞分化阶段的不同而不同。我们的单DSB分析方法对于确定遗传和环境改变对多能细胞及其分化后代修复精度的影响具有广泛的实用价值。
Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.
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影响因子：
46.9
作者：
Hockemeyer, Dirk;Soldner, Frank;Beard, Caroline;Gao, Qing;Mitalipova, Maisam;DeKelver, Russell C.;Katibah, George E.;Amora, Ranier;Boydston, Elizabeth A.;Zeitler, Bryan;Meng, Xiangdong;Miller, Jeffrey C.;Zhang, Lei;Rebar, Edward J.;Gregory, Philip D.;Urnov, Fyodor D.;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
影响因子：
5.2
作者：
Gerrard, L;Rodgers, L;Cui, W
通讯作者：
Cui, W
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3.8
作者：
Banuelos, C. A.;Banath, J. P.;Olive, P. L.
通讯作者：
Olive, P. L.
影响因子：
64.8
作者：
Esashi, F;Christ, N;West, SC
通讯作者：
West, SC
影响因子：
11.4
作者：
Boskovic, J;Rivera-Calzada, A;Llorca, O
通讯作者：
Llorca, O