Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells
Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells
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DOI：
10.1016/j.celrep.2015.06.051
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发表时间：
2015-07-28
期刊：
影响因子：
8.8
通讯作者：
Buehler, Marc
中科院分区：
文献类型：
--
作者：
Flemr, Matyas;Buehler, Marc
工程核酸酶，如CRISPR/Cas9或转录激活因子样核酸酶(TALENS)诱导双链DNA断裂(DSB)，通过同源重组(HR)刺激外源DNA片段的敲击。然而，到目前为止所报道的敲门效率还不允许更复杂的体外基因组修改，例如，在两个不同的基因组位置同时整合DNA片段。我们开发了一种报告系统，以丰富工程核酸酶辅助的HR事件的细胞。将该系统应用于小鼠胚胎干细胞，实现了Cre重组酶介导的诱导性基因敲除的两个loxP位点的一步双等位无缝整合，以及高效的双等位基因标记。我们的方法大大减少了条件基因敲除mESC生成所需的时间和资源。
Induction of double-strand DNA breaks (DSBs) by engineered nucleases, such as CRISPR/Cas9 or transcription activator-like effector nucleases (TALENs), stimulates knockin of exogenous DNA fragments via homologous recombination (HR). However, the knockin efficiencies reported so far have not allowed more complex in vitro genome modifications such as, for instance, simultaneous integration of a DNA fragment at two distinct genomic sites. We developed a reporter system to enrich for cells with engineered nuclease-assisted HR events. Using this system in mouse embryonic stem cells (mESCs), we achieve single-step biallelic and seamless integration of two loxP sites for Cre recombinase-mediated inducible gene knockout, as well as biallelic endogenous gene tagging with high efficiency. Our approach reduces the time and resources required for conditional knockout mESC generation dramatically.