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Genome Research
Year: 2010  |  Volume: 20  |  Issue: 8  |  Page No.: 1133 - 1142

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

R. C DeKelver, V. M Choi, E. A Moehle, D. E Paschon, D Hockemeyer, S. H Meijsing, Y Sancak, X Cui, E. J Steine, J. C Miller, P Tam, V. V Bartsevich, X Meng, I Rupniewski, S. M Gopalan, H. C Sun, K. J Pitz, J. M Rock, L Zhang, G. D Davis, E. J Rebar, I. M Cheeseman, K. R Yamamoto, D. M Sabatini, R Jaenisch, P. D Gregory and F. D. Urnov    

Abstract:

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.

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