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

Dirk Hockemeyer; Frank Soldner; Caroline Beard; Qing Gao; Maisam Mitalipova; Russell C. Dekelver; George E. Katibah; Ranier Amora; Elizabeth A. Boydston; Bryan Zeitler; Xiangdong Meng; Jeffrey C. Miller; Lei Zhang; Edward J. Rebar; Philip D. Gregory; Fyodor D. Urnov; Rudolf Jaenisch

doi:10.1038/nbt.1562

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

Dirk Hockemeyer, Frank Soldner, Caroline Beard, Qing Gao, Maisam Mitalipova, Russell C. Dekelver, George E. Katibah, Ranier Amora, Elizabeth A. Boydston, Bryan Zeitler, Xiangdong Meng, Jeffrey C. Miller, Lei Zhang, Edward J. Rebar, Philip D. Gregory, Fyodor D. Urnov, Rudolf Jaenisch

Research output: Contribution to journal › Article › peer-review

877 Scopus citations

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.

Original language	English (US)
Pages (from-to)	851-857
Number of pages	7
Journal	Nature biotechnology
Volume	27
Issue number	9
DOIs	https://doi.org/10.1038/nbt.1562
State	Published - Sep 2009
Externally published	Yes

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology
Molecular Medicine
Biomedical Engineering

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10.1038/nbt.1562

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Hockemeyer, D., Soldner, F., Beard, C., Gao, Q., Mitalipova, M., Dekelver, R. C., Katibah, G. E., Amora, R., Boydston, E. A., Zeitler, B., Meng, X., Miller, J. C., Zhang, L., Rebar, E. J., Gregory, P. D., Urnov, F. D., & Jaenisch, R. (2009). Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nature biotechnology, 27(9), 851-857. https://doi.org/10.1038/nbt.1562

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 2009, 'Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases', Nature biotechnology, vol. 27, no. 9, pp. 851-857. https://doi.org/10.1038/nbt.1562

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title = "Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases",

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.",

author = "Dirk Hockemeyer and Frank Soldner and Caroline Beard and Qing Gao and Maisam Mitalipova and Dekelver, {Russell C.} and Katibah, {George E.} and Ranier Amora and Boydston, {Elizabeth A.} and Bryan Zeitler and Xiangdong Meng and Miller, {Jeffrey C.} and Lei Zhang and Rebar, {Edward J.} and Gregory, {Philip D.} and Urnov, {Fyodor D.} and Rudolf Jaenisch",

note = "Funding Information: We thank R. Alagappan, P. Xu and E. Cook for technical support and J. Dausman, R. Flannery and D. Fu for their help with animal husbandry and processing of teratomas. We thank all the members of the Jaenisch laboratory for helpful discussions and comments on the manuscript. D.H. is a Merck Fellow of the Life Science Research Foundation. R.J. was supported by US National Institutes of Health grants R37-CA084198, RO1-CA087869 and RO1-HD045022: and by the Howard Hughes Medical Institute. Requests for ZFNs should be directed to F.D.U. (furnov@sangamo.com).",

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AU - Mitalipova, Maisam

AU - Dekelver, Russell C.

AU - Katibah, George E.

AU - Amora, Ranier

AU - Boydston, Elizabeth A.

AU - Zeitler, Bryan

AU - Meng, Xiangdong

AU - Miller, Jeffrey C.

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AU - Rebar, Edward J.

AU - Gregory, Philip D.

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

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