Skip to main content

Gene Transfer Via Nucleofection Into Adult and Embryonic Stem Cells

  • Protocol
Stem Cell Assays

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 407))

Summary

The use of embryonic and adult stem cells as therapeutic agents is gaining momentum. A major impediment in the use of stem cells for genetic disorders is their ability to undergo genetic modification. The recognition of various site-specific integration methods open up a new avenue for gene therapy in stem cells. However, this necessitates efficient delivery of DNA molecule into cells. Most commercially used liposome-mediated transfection reagents are toxic or work poorly with stem cells. Electroporation, while effective in transfecting stem cells, is rather harsh and leads to excessive cell death. Nucleofection, a technology by Amaxa, uses a combination of electric pulse in an appropriate media, which decreases the toxicity and promotes efficient transfection of stem cells. Various types of adult and embryonic stem cells can be successfully transfected using this method, as described in this chapter.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Bonetta, L. (2005) The inside scoop-evaluating gene delivery methods. Nat Methods, 2, 875–883.

    Article  CAS  Google Scholar 

  2. Lakshmipathy, U., Pelacho, B., Sudo, K., Linehan, J.L., Coucouvanis, E., Kaufman, D.S. and Verfaillie, C.M. (2004) Efficient transfection of embryonic and adult stem cells. Stem Cells, 22, 531–543.

    Article  PubMed  Google Scholar 

  3. Lakshmipathy U., Hammer, L. Verfaillie C. (2004) A nonviral gene transfer method for transfecting multipotent adult progenitor cells (MAPC). Gene Therapy and Regulation, 2, 301–312.

    Article  CAS  Google Scholar 

  4. Liu, Y.P., Dovzhenko, O.V., Garthwaite, M.A., Dambaeva, S.V., Durning, M., Pollastrini, L.M. and Golos, T.G. (2004) Maintenance of pluripotency in human embryonic stem cells stably over-expressing enhanced green fluorescent protein. Stem Cells Dev, 13, 636–645.

    Article  CAS  PubMed  Google Scholar 

  5. Nowling, T., Desler, M., Kuszynski, C. and Rizzino, A. (2002) Transfection of embryonal carcinoma cells at high efficiency using liposome-mediated transfection. Mol Reprod Dev, 63, 309–317.

    Article  CAS  PubMed  Google Scholar 

  6. Ren, C.P., Zhao, M., Shan, W.J., Yang, X.Y., Yin, Z.H., Jiang, X.J., Zhang, H.B. and Yao, K.T. (2005) Establishment of human embryonic stem cell line stably expressing Epstein-Barr virus-encoded nuclear antigen 1. Acta Biochim Biophys Sin (Shanghai), 37, 68–73.

    Article  CAS  Google Scholar 

  7. Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz-Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M. et al. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 418, 41–49.

    Article  CAS  PubMed  Google Scholar 

  8. Aluigi, M., Fogli, M., Curti, A., Isidori, A., Gruppioni, E., Chiodoni, C., Colombo, M.P., Versura, P., D’Errico-Grigioni, A., Ferri, E. et al. (2006) Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. Stem Cells, 24, 454–461.

    Article  PubMed  Google Scholar 

  9. Haleem-Smith, H., Derfoul, A., Okafor, C., Tuli, R., Olsen, D., Hall, D.J. and Tuan, R.S. (2005) Optimization of high-efficiency transfection of adult human mesenchymal stem cells in vitro. Mol Biotechnol, 30, 9–20.

    Article  CAS  PubMed  Google Scholar 

  10. Kobayashi, N., Rivas-Carrillo, J.D., Soto-Gutierrez, A., Fukazawa, T., Chen, Y., Navarro-Alvarez, N. and Tanaka, N. (2005) Gene delivery to embryonic stem cells. Birth Defects Res C Embryo Today, 75, 10–18.

    Article  CAS  PubMed  Google Scholar 

  11. Lorenz, P., Harnack, U. and Morgenstern, R. (2004) Efficient gene transfer into murine embryonic stem cells by nucleofection. Biotechnol Lett, 26, 1589–92.

    Article  CAS  PubMed  Google Scholar 

  12. Quenneville, S.P., Chapdelaine, P., Rousseau, J., Beaulieu, J., Caron, N.J., Skuk, D., Mills, P., Olivares, E.C., Calos, M.P. and Tremblay, J.P. (2004) Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts. Mol Ther, 10, 679–87.

    Article  CAS  PubMed  Google Scholar 

  13. Siemen, H., Nix, M., Endl, E., Koch, P., Itskovitz-Eldor, J. and Brustle, O. (2005) Nucleofection of human embryonic stem cells. Stem Cells Dev, 14, 378–83.

    Article  CAS  PubMed  Google Scholar 

  14. Spangrude, G.J., Klein, J., Heimfeld, S., Aihara, Y. and Weissman, I.L. (1989) Two monoclonal antibodies identify thymic-repopulating cells in mouse bone marrow. J Immunol, 142, 425–30.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2007 Humana Press

About this protocol

Cite this protocol

Lakshmipathy, U., Buckley, S., Verfaillie, C. (2007). Gene Transfer Via Nucleofection Into Adult and Embryonic Stem Cells. In: Vemuri, M.C. (eds) Stem Cell Assays. Methods in Molecular Biology™, vol 407. Humana Press. https://doi.org/10.1007/978-1-59745-536-7_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-536-7_9

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-744-0

  • Online ISBN: 978-1-59745-536-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics