Sangamo BioSciences, Inc., Point Richmond Tech Center, Richmond, California 94804, USA.
Genome Res. 2010 Aug;20(8):1133-42. doi: 10.1101/gr.106773.110. Epub 2010 May 27.
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 AAVS1. 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 50 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.
在模式生物中，同基因设置是常规操作，但对于人类细胞的遗传实验仍然难以实现。我们描述了使用设计的锌指核酸酶 (ZFN) 进行高效转基因，而无需在 PPP1R12C 基因（一个称为 AAVS1 的“安全港”基因座）中进行药物选择。ZFN 可在瞬时转染转化和原代人类细胞（包括成纤维细胞和 hES 细胞）后，以高达 15%的频率实现靶向转基因。当将其添加到该基因座时，转基因，如 shRNA 的表达盒、小分子反应性 cDNA 表达盒和报告基因构建体，在超过 50 个细胞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，这种方法允许在实质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、蛋白质组学和调控 DNA 分析的真正同基因设置。
