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)).

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

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

  2. 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).

  3. 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).

  4. 4

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

  5. 5

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

  6. 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).

  7. 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).

  8. 8

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

  9. 9

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

  10. 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).

  11. 11

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

  12. 12

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

  13. 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).

  14. 14

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

  15. 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).

  16. 16

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

  17. 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).

  18. 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).

  19. 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).

  20. 20

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

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.

Author information

Correspondence to James A. Thomson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading