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          Institute: MPI für molekulare Genetik     Collection: Department of Computational Molecular Biology     Display Documents



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ID: 536307.0, MPI für molekulare Genetik / Department of Computational Molecular Biology
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
Authors:DeKelver, R. C.; Choi, V. M.; Moehle, E. A.; Paschon, D. E.; Hockemeyer, D.; Meijsing, S. H.; Sancak, Y.; Cui, X.; Steine, E. J.; Miller, J. C.; Tam, P.; Bartsevich, V. V.; Meng, X.; Rupniewski, I.; Gopalan, S. M.; Sun, H. C.; Pitz, K. J.; Rock, J. M.; Zhang, L.; Davis, G. D.; Rebar, E. J.; Cheeseman, I. M.; Yamamoto, K. R.; Sabatini, D. M.; Jaenisch, R.; Gregory, P. D.; Urnov, F. D.
Language:English
Date of Publication (YYYY-MM-DD):2010-08
Title of Journal:Genome Research
Journal Abbrev.:Genome Res
Volume:20
Issue / Number:8
Start Page:1133
End Page:1142
Copyright:© 2010 by Cold Spring Harbor Laboratory Press
Review Status:not specified
Audience:Not Specified
Abstract / Description: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.
External Publication Status:published
Document Type:Article
Communicated by:Martin Vingron
Affiliations:MPI für molekulare Genetik
External Affiliations:Sangamo BioSciences, Inc., Point Richmond Tech Center, Richmond, California 94804, USA
The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158, USA
Sigma-Aldrich Research Biotechnology, St. Louis, Missouri 63103, USA
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
Identifiers:ISSN:1549-5469 (Electronic) [ID No:1]
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