Journal
GENOME RESEARCH
Volume 20, Issue 8, Pages 1133-1142Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1101/gr.106773.110
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Funding
- US National Institutes of Health [R37-CA084198, RO1-CA087869, RO1-HD045022]
- Howard Hughes Medical Institute
- NIH [R01 CA103866, AI47389]
- Merck and Company
- Department of Defense [W81XWH-07-0448]
- W M Keck Foundation
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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.
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