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A method for genetic modification of human embryonic stem cells using electroporation

Nature Protocols volume 2, pages 792–796 (2007)Cite this article

Abstract

The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.

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Figure 1: Photomicrographs of HESCs in organ culture.
Figure 2: Morphology of HESCs expanded in bulk culture, following electroporation and during selection in G418.

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Acknowledgements

We thank Elizabeth Ng for her valuable contribution to the development of the organ culture protocol described in this report.

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Author notes

  1. Magdaline Costa and Mirella Dottori: These authors contributed equally to this work.

Authors and Affiliations

  1. Monash Immunology and Stem Cell Laboratories, STRIP 1, West Ring Road, Monash University Campus, Clayton, 3800, Victoria, Australia

    Magdaline Costa, Koula Sourris, Tanya Hatzistavrou, Richard Davis, Lisa Azzola, Steven Jackson, Sue Mei Lim, Andrew G Elefanty & Edouard G Stanley

  2. Monash Institute of Medical Research and the Australian Stem Cell Centre, Clayton, Victoria, Australia

    Mirella Dottori, Pegah Jamshidi & Martin Pera

  3. Center for Stem Cell and Regenerative Medicine, Keck School of Medicine, University of Southern California, California, 90089–9112, USA

    Martin Pera

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  1. Magdaline Costa
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  3. Koula Sourris
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  4. Pegah Jamshidi
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Correspondence to Edouard G Stanley.

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Costa, M., Dottori, M., Sourris, K. et al. A method for genetic modification of human embryonic stem cells using electroporation. Nat Protoc 2, 792–796 (2007). https://doi.org/10.1038/nprot.2007.105

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