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PMID: 20508142 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

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.

Genome research ·Vol. 20 ·No. 8 ·2010-08-00 ·Pages 1133-42

DeKelver RC, Choi VM, Moehle EA, Paschon DE, Hockemeyer D, Meijsing SH, Sancak Y, Cui X, Steine EJ, Miller JC, Tam P, Bartsevich VV, Meng X, Rupniewski I, Gopalan SM, Sun HC, Pitz KJ, Rock JM, Zhang L, Davis GD, Rebar EJ, Cheeseman IM, Yamamoto KR, Sabatini DM, Jaenisch R, Gregory PD, Urnov FD

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.

MeSH Terms
Base Sequence Cell Line Endonucleases/genetics Gene Transfer Techniques Genetic Loci Genome, Human Genomics/methods Humans Molecular Sequence Data Proteomics/methods Regulatory Sequences, Nucleic Acid/genetics Zinc Fingers/genetics
Chemicals
Endonucleases
Authors & Affiliations
27 authors, click to expand affiliations / ORCID
DeKelver Russell C
Sangamo BioSciences, Inc., Point Richmond Tech Center, Richmond, California 94804, USA.
Choi Vivian M
Moehle Erica A
Paschon David E
Hockemeyer Dirk
Meijsing Sebastiaan H
Sancak Yasemin
Cui Xiaoxia
Steine Eveline J
Miller Jeffrey C
Tam Phillip
Bartsevich Victor V
Meng Xiangdong
Rupniewski Igor
Gopalan Sunita M
Sun Helena C
Pitz Kathleen J
Rock Jeremy M
Zhang Lei
Davis Gregory D
Rebar Edward J
Cheeseman Iain M
Yamamoto Keith R
Sabatini David M
Jaenisch Rudolf
Gregory Philip D
Urnov Fyodor D
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Article Info
Journal
Genome research
Abbr.
Genome Res
ISSN
1549-5469
DOI
10.1101/gr.106773.110
Published
2010-08-00
Epub
2010-00-27
Pages
1133-42
Language
English
Region
United States
NLM ID
9518021
PMCID
PMC2909576
Subset
IM
Grants
NCI NIH HHS · R01-CA087869 · United States
Howard Hughes Medical Institute · United States
NICHD NIH HHS · R01-HD045022 · United States
NCI NIH HHS · R37-CA084198 · United States
NICHD NIH HHS · R37 HD045022 · United States
NIAID NIH HHS · R37 AI047389 · United States
NCI NIH HHS · R37 CA084198 · United States
NCI NIH HHS · R01 CA129105 · United States
NCI NIH HHS · R01 CA129105-04 · United States
NCI NIH HHS · R01 CA087869 · United States
NIAID NIH HHS · AI47389 · United States
NIAID NIH HHS · R01 AI047389 · United States
NCI NIH HHS · R01 CA103866 · United States
NICHD NIH HHS · R01 HD045022 · United States
NCI NIH HHS · R01 CA084198 · United States
External Links
PubMed source DOI: 10.1101/gr.106773.110 PMC Full Text View journal details
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