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
The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
Nat Biotechnol. 2009 Sep;27(9):851-7. doi: 10.1038/nbt.1562. Epub 2009 Aug 13.
Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
要实现人类胚胎干细胞（hESCs）和诱导多能干细胞（hiPSCs）的全部潜能，需要高效的基因改造方法。然而，生成细胞类型特异性谱系报告基因的技术，以及通过基因打靶来破坏、修复或过表达基因的可靠工具，充其量效率都很低，因此未被常规使用。在此，我们报告了使用锌指核酸酶（ZFN）介导的基因组编辑对人类多能细胞中的三个基因进行高效靶向。首先，使用针对OCT4（POU5F1）基因座的ZFN，我们生成了OCT4-eGFP报告细胞，以监测hESCs的多能状态。其次，我们将一个转基因插入AAVS1基因座，以在hESCs中生成一个强大的药物诱导过表达系统。最后，我们靶向PITX3基因，证明ZFN可用于通过靶向hESCs和hiPSCs中未表达的基因来生成报告细胞。
