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PMID: 19680244 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.

Nature biotechnology ·Vol. 27 ·No. 9 ·2009-09-00 ·Pages 851-7

Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R

Abstract

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.

MeSH Terms
Cell Line Deoxyribonucleases/genetics,metabolism Embryonic Stem Cells/physiology Gene Expression Gene Silencing Gene Targeting/methods Green Fluorescent Proteins/genetics,metabolism Homeodomain Proteins/genetics,metabolism Humans Immunohistochemistry Octamer Transcription Factor-3/genetics,metabolism Pluripotent Stem Cells/physiology Recombinant Fusion Proteins/genetics,metabolism Transcription Factors/genetics,metabolism Zinc Fingers/physiology
Chemicals
Homeodomain Proteins Octamer Transcription Factor-3 POU5F1 protein, human Recombinant Fusion Proteins Transcription Factors enhanced green fluorescent protein homeobox protein PITX3 Green Fluorescent Proteins Deoxyribonucleases
Authors & Affiliations
17 authors, click to expand affiliations / ORCID
Hockemeyer Dirk
The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
Soldner Frank
Beard Caroline
Gao Qing
Mitalipova Maisam
DeKelver Russell C
Katibah George E
Amora Ranier
Boydston Elizabeth A
Zeitler Bryan
Meng Xiangdong
Miller Jeffrey C
Zhang Lei
Rebar Edward J
Gregory Philip D
Urnov Fyodor D
Jaenisch Rudolf
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Article Info
Journal
Nature biotechnology
Abbr.
Nat Biotechnol
ISSN
1546-1696
DOI
10.1038/nbt.1562
Published
2009-09-00
Epub
2009-00-13
Pages
851-7
Language
English
Region
United States
NLM ID
9604648
PMCID
PMC4142824
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
NCI NIH HHS · R37 CA084198 · United States
NCI NIH HHS · R01 CA087869 · United States
NICHD NIH HHS · R01 HD045022 · United States
NCI NIH HHS · R01 CA084198 · United States
External Links
PubMed source DOI: 10.1038/nbt.1562 PMC Full Text View journal details
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