Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells
Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells
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DOI：
10.1093/nar/23.24.5012
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发表时间：
1995-12-25
影响因子：
14.9
通讯作者：
Jasin, M
中科院分区：
文献类型：
--
作者：
Smih, F;Rouet, P;Jasin, M
双链断裂（DSB）是酵母、果蝇和秀丽隐杆线虫染色体DNA的重组损伤。最近在哺乳动物细胞中利用I-Scel内切核酸酶的研究已经证明，在一些永生化细胞系中，染色体DNA中的DSB也是重组的。我们现在已经测试了胚胎干（ES）细胞，一种经常用于基因靶向研究的非转化小鼠细胞系。我们发现，由I-Scel引入的DSB刺激在可选择的neo基因座处的基因靶向至少50倍。通过瞬时表达I-SceI内切核酸酶实现增强的靶向水平。在97%的靶向克隆中，转染的同源片段中的单碱基对多态性被并入靶基因座。靶向位点的分析表明，大多数同源重组事件是“双侧”的，与以前在3 T3细胞中的研究相反，其中“单侧”同源事件占主导地位。因此，ES细胞可能比培养中的其他细胞更忠实地将同源片段整合到其基因组中。
Double-strand breaks (DSBs) are recombinogenic lesions in chromosomal DNA in yeast, Drosophila and Caenorhabditis elegans. Recent studies in mammalian cells utilizing the I-Scel endonuclease have demonstrated that in some immortalized cell lines DSBs in chromosomal DNA are also recombinogenic, We have now tested embryonic stem (ES) cells, a non-transformed mouse cell line frequently used in gene targeting studies, We find that a DSB introduced by I-Scel stimulates gene targeting at a selectable neo locus at least 50-fold. The enhanced level of targeting is achieved by transient expression of the I-Scel endonuclease. In 97% of targeted clones a single base pair polymorphism in the transfected homologous fragment was incorporated into the target locus. Analysis of the targeted locus demonstrated that most of the homologous recombination events were 'two-sided', in contrast to previous studies in 3T3 cells in which 'one-sided' homologous events predominated. Thus ES cells may be more faithful in incorporating homologous fragments into their genome than other cells in culture.