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Homologous recombination in human embryonic stem cells

Abstract

Homologous recombination applied to mouse embryonic stem (ES) cells has revolutionized the study of gene function in mammals1,2,3,4. Although most often used to generate knockout mice, homologous recombination has also been applied in mouse ES cells allowed to differentiate in vitro. Homologous recombination is an essential technique if human ES cells5 are to fulfill their promise as a basic research tool. It also has important implications for ES cell–based transplantation and gene therapies. Significant differences between mouse and human ES cells have hampered the development of homologous recombination in human ES cells. High, stable transfection efficiencies in human ES cells have been difficult to achieve, and, in particular, electroporation protocols established for mouse ES cells work poorly in human ES cells6. Also, in contrast to their murine counterparts, human ES cells cannot be cloned efficiently from single cells, making it difficult to screen for rare recombination events7. Here we report an electroporation approach, based on the physical characteristics of human ES cells, that we used to successfully target HPRT1, the gene encoding hypoxanthine phosphoribosyltransferase-1 (HPRT1), and POU5F1, the gene encoding octamer-binding transcription factor 4 (Oct4; also known as POU domain, class 5, transcription factor 1 (POU5F1)).

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Figure 1: Targeted deletion of the last three exons of the HPRT1 gene.
Figure 2: Targeting of an IRES-EGFP-IRES-neo cassette into the 3′ UTR of the gene POU5F1, which encodes Oct4.

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References

  1. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  2. Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638 (1981).

    Article  CAS  Google Scholar 

  3. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. & Kucherlapati, R.S. Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination. Nature 317, 230–234 (1985).

    Article  CAS  Google Scholar 

  4. Thomas, K.R. & Capecchi, M.R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512 (1987).

    Article  CAS  Google Scholar 

  5. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  Google Scholar 

  6. Eiges, R. et al. Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr. Biol. 11, 514–518 (2001).

    Article  CAS  Google Scholar 

  7. Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278 (2000).

    Article  CAS  Google Scholar 

  8. Albertini, R.J. HPRT mutations in humans: biomarkers for mechanistic studies. Mutat. Res. 489, 1–16 (2001).

    Article  CAS  Google Scholar 

  9. Doetschman, T. et al. Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330, 576–578 (1987).

    Article  CAS  Google Scholar 

  10. Vasquez, K.M., Marburger, K., Intody, Z. & Wilson, J.H. Manipulating the mammalian genome by homologous recombination. Proc. Natl. Acad. Sci. USA 98, 8403–8410 (2001).

    Article  CAS  Google Scholar 

  11. Fehling, H.J. et al. MHC class I expression in mice lacking the proteasome subunit LMP-7. Science 265, 1234–1237 (1994).

    Article  CAS  Google Scholar 

  12. Muller, M. et al. Selection of ventricular-like cardiomyocytes from ES cells in vitro. FASEB J. 14, 2540–2548 (2000).

    Article  CAS  Google Scholar 

  13. Mountford, P., Nichols, J., Zevnik, B., O'Brien, C. & Smith, A. Maintenance of pluripotential embryonic stem cells by stem cell selection. Reprod. Fertil. Dev. 10, 527–533 (1998).

    Article  CAS  Google Scholar 

  14. Pesce, M. & Scholer, H.R. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19, 271–278 (2001).

    Article  CAS  Google Scholar 

  15. Niwa, H., Miyazaki, J. & Smith, A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).

    Article  CAS  Google Scholar 

  16. Mountford, P. et al. Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc. Natl. Acad. Sci. USA 91, 4303–4307 (1994).

    Article  CAS  Google Scholar 

  17. Rideout, W.M., 3rd, Hochedlinger, K., Kyba, M., Daley, G.Q. & Jaenisch, R. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109, 17–27 (2002).

    Article  CAS  Google Scholar 

  18. Finger, S., Heavens, R.P., Sirinathsinghji, D.J., Kuehn, M.R. & Dunnett, S.B. Behavioral and neurochemical evaluation of a transgenic mouse model of Lesch-Nyhan syndrome. J. Neurol. Sci. 86, 203–213 (1988).

    Article  CAS  Google Scholar 

  19. Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O. & Thomson, J.A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133 (2001).

    Article  CAS  Google Scholar 

  20. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Henry Yuen for his gift to the University of Wisconsin Foundation that supports this work. We thank S. Witowski, J. Antosiewicz, K. Murphy, and O. Weber for technical assistance and H. J. Fehling for many valuable discussions. This is Publication 41-013 of the Wisconsin Regional Primate Research Center.

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Correspondence to James A. Thomson.

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Zwaka, T., Thomson, J. Homologous recombination in human embryonic stem cells. Nat Biotechnol 21, 319–321 (2003). https://doi.org/10.1038/nbt788

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