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Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase

Nature Methods volume 3, pages 461–467 (2006)Cite this article

Abstract

The biomedical application of human embryonic stem (hES) cells will increasingly depend on the availability of technologies for highly controlled genetic modification. In mouse genetics, conditional mutagenesis using site-specific recombinases has become an invaluable tool for gain- and loss-of-function studies. Here we report highly efficient Cre-mediated recombination of a chromosomally integrated loxP-modified allele in hES cells and hES cell–derived neural precursors by protein transduction. Recombinant modified Cre recombinase protein translocates into the cytoplasm and nucleus of hES cells and subsequently induces recombination in virtually 100% of the cells. Cre-transduced hES cells maintain the expression of pluripotency markers as well as the capability of differentiating into derivatives of all three germ layers in vitro and in vivo. We expect this technology to provide an important technical basis for analyzing complex genetic networks underlying human development as well as generating highly purified, transplantable hES cell–derived cells for regenerative medicine.

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Figure 1: Cre double fluorescence reporter construct FDR.
Figure 2: Recombinant modified Cre protein translocates into hES cells and induces recombination of loxP-modified alleles.
Figure 3: Quantification of Cre protein transduction and subsequent recombination in hES-FDR1 cells.
Figure 4: Cre-treated hES cells retain normal proliferation and pluripotency.
Figure 5: Cre protein transduction into hES cell-derived neural precursor cells.

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Acknowledgements

We thank J. Itskovitz-Eldor for providing the H9.2 cell line and E. Kolossov for providing materials. Also, we thank S. Terstegge and C. Benzing for the initial set up of the hES culture and M. Peitz, E. Endl, L. Heukamp as well as members of the Stem Cell Engineering Group for support and valuable discussions. We thank M. Segschneider, A. Leinhaas and members of the histology laboratory of the Institute of Neuropathology, University of Bonn, for technical assistance with embryoid body staining and teratoma analysis. This work was supported by grants from the Stem Cell Network North Rhine Westphalia (400 004 03), the European Union (LSHB-CT-20003-503005; EUROSTEMCELL), the Volkswagen Foundation (Az I/77864), and grants from the Deutsche Forschungsgemeinschaft (BR 1337/3-2) and the Hertie Foundation.

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Author notes

  1. Henrike Siemen

    Present address: Center for Reproductive Science, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, California, 94143, USA

  2. Lars Nolden and Frank Edenhofer: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn and Hertie Foundation, Sigmund Freud Street 25, Bonn, 53105, Germany

    Lars Nolden, Frank Edenhofer, Simone Haupt, Philipp Koch, Henrike Siemen & Oliver Brüstle

  2. Stem Cell Engineering Group at the Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn and Hertie Foundation, Sigmund Freud Street 25, Bonn, 53105, Germany

    Frank Edenhofer & Simone Haupt

  3. Institute for Genetics, University of Cologne, Zülpicher Street 47, Cologne, 50674, Germany

    F Thomas Wunderlich

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  1. Lars Nolden
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Corresponding author

Correspondence to Frank Edenhofer.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Validation of the FDR construct. (PDF 351 kb)

Supplementary Fig. 2

Native eGFP fluorescence of Cre-transduced hES-FDR1 cells. (PDF 344 kb)

Supplementary Fig. 3

Maintenance of pluripotency of Cre-transduced hES-FDR1 cells. (PDF 1192 kb)

Supplementary Table 1

Construction of pFDR Cre reporter plasmid. (PDF 140 kb)

Supplementary Methods (PDF 67 kb)

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Nolden, L., Edenhofer, F., Haupt, S. et al. Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase. Nat Methods 3, 461–467 (2006). https://doi.org/10.1038/nmeth884

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