Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome
Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome
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DOI：
10.1101/gr.106773.110
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发表时间：
2010-08-01
期刊：
影响因子：
7
通讯作者：
Urnov, Fyodor D.
中科院分区：
文献类型：
--
作者：
DeKelver, Russell C.;Choi, Vivian M.;Urnov, Fyodor D.
等基因设置在模式生物中是常规的，但在人类细胞的遗传实验中仍然难以捉摸。我们描述了使用设计锌指核酸酶（ZFNs）进行高效转基因，无需药物选择进入PPP1R12C基因，这是一个被称为AAVSI的“安全港”位点。瞬时转染转化和原代人细胞（包括成纤维细胞和he细胞）后，ZFNs能够以高达15%的频率进行靶向转基因。当添加到该位点时，shrna表达盒、小分子响应性cDNA表达盒和报告基因构建等转基因在SO细胞世代中表现出一致的表达和持续的功能。通过避免随机整合和药物选择，该方法允许在基本上任何转化的人类细胞类型和原代细胞中进行高通量功能基因组学，蛋白质组学和调节性DNA分析的真正等基因设置。
Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVSI. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over SO cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.