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

Nature Biotechnology volume 27, pages 851–857 (2009)Cite this article

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.

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Figure 1: Targeting of OCT4 in hESCs using ZFNs.
Figure 2: Targeting of the AAVS1 locus using ZFNs.
Figure 3: Targeting of PITX3 in hESCs and hiPSCs using ZFNs.

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Acknowledgements

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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Author notes

  1. Dirk Hockemeyer and Frank Soldner: These authors contributed equally to this work.

Authors and Affiliations

  1. The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Dirk Hockemeyer, Frank Soldner, Caroline Beard, Qing Gao, Maisam Mitalipova & Rudolf Jaenisch

  2. Sangamo BioSciences, Inc., Richmond, California, USA

    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

  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Rudolf Jaenisch

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  1. Dirk Hockemeyer
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Contributions

D.H., F.S. and R.J. designed the experiments and wrote the paper. C.B. provided assistance with construct design and Southern blot analysis. Q.G. analyzed all teratomas. M.M. provided hESCs. J.C.M. and L.Z. designed the ZFNs, which were assembled and tested by R.C.D., G.E.K. and R.A. X.M. performed the SELEX experiments. E.A.B. and B.Z. genotyped ZFN-edited clones for off-target effects. F.D.U., E.J.R. and P.D.G. supervised the ZFN design, characterization and off-target analysis and helped analyze the data and write the paper. D.H. and F.S. performed all other experiments.

Corresponding author

Correspondence to Rudolf Jaenisch.

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Competing interests

R.J. is an adviser to Stemgen and a cofounder of Fate Therapeutics. R.C.D., G.E.K., R.A., B.Z., X.M., J.C.M., L.Z., E.J.R., P.D.G. and F.D.U. are full-time employees of Sangamo BioSciences, Inc.

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Hockemeyer, D., Soldner, F., Beard, C. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27, 851–857 (2009). https://doi.org/10.1038/nbt.1562

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