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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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.
The authors declare no competing financial interests.
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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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