Skip to main content

Production of Multicistronic HIV-1 Based Lentiviral Vectors

  • Protocol
Viral Applications of Green Fluorescent Protein

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 515))

Summary

In the last decade lentiviral gene transfer vectors have gained significant place both in basic science and gene therapy applications. A number of gene transfer applications would benefit from vectors capable of expressing multiple genes. This chapter focuses on production of bicistronic and tricistronic lentiviral vectors based on the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. Multigene vectors produced high titer viral particles and were able to simultaneously express two or three transgenes in transduced cells. The level of expression of individual transgenes varied depending on the transgene itself, its position within the construct, the total number of transgenes expressed, the strategy used for multigene expression, and the number of copies of proviral insertions.

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight 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. Ngoi, S.M., Chien, A.C. and Lee, C.G. Exploiting internal ribosome entry sites in gene therapy vector design, Curr. Gene Ther., 4: 15–31, 2004.

    Article  PubMed  CAS  Google Scholar 

  2. Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E. and Hayakawa, T. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector, Mol. Ther., 1: 376–382, 2000.

    Article  PubMed  CAS  Google Scholar 

  3. Zhou, Y., Aran, J., Gottesman, M.M. and Pastan, I. Co-expression of human adenosine deaminase and multidrug resistance using a bicistronic retroviral vector, Hum. Gene Ther., 9: 287–293, 1998.

    Article  CAS  Google Scholar 

  4. Furler, S., Paterna, J.C., Weibel, M. and Bueler, H. Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons, Gene Ther., 8: 864–873, 2001.

    Article  PubMed  CAS  Google Scholar 

  5. de Felipe, P., Martin, V., Cortes, M.L., Ryan, M. and Izquierdo, M. Use of the 2A sequence from foot-and-mouth disease virus in the generation of retroviral vectors for gene therapy, Gene Ther., 6: 198–208, 1999.

    Article  PubMed  CAS  Google Scholar 

  6. Klump, H., Schiedlmeier, B., Vogt, B., Ryan, M., Ostertag, W. and Baum, C. Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy, Gene Ther., 8: 811–817, 2001.

    Article  PubMed  CAS  Google Scholar 

  7. Stripecke, R., Cardoso, A.A., Pepper, K.A., Skelton, D.C., Yu, X.J., Mascarenhas, L., Weinberg, K.I., Nadler, L.M. and Kohn, D.B. Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage-colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses, Blood, 96: 1317–1326, 2000.

    PubMed  CAS  Google Scholar 

  8. Reiser, J., Lai, Z., Zhang, X.Y. and Brady, R.O. Development of multigene and regulated lentivirus vectors, J. Virol., 74: 10589–10599, 2000.

    Article  PubMed  CAS  Google Scholar 

  9. Yu, X., Zhan, X., D’Costa, J., Tanavde, V.M., Ye, Z., Peng, T., Malehorn, M.T., Yang, X., Civin, C.I. and Cheng, L. Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells, Mol. Ther., 7: 827–838, 2003.

    Article  PubMed  CAS  Google Scholar 

  10. Zhu, Y. and Planelles, V. A multigene lentiviral vector system based on differential splicing, Methods Mol. Med., 76: 433–448, 2003.

    PubMed  CAS  Google Scholar 

  11. Chinnasamy, D., Fairbairn, L.J., Neuenfeldt, J., Treisman, J.S., Hanson, J.P., Jr., Margison, G.P. and Chinnasamy, N. Lentivirus-mediated expression of mutant MGMTP140K protects human CD34+ cells against the combined toxicity of O6-benzylguanine and 1,3-bis(2-chloroethyl)-nitrosourea or temozolomide, Hum. Gene Ther., 15: 758–769, 2004.

    Article  PubMed  CAS  Google Scholar 

  12. Chinnasamy, D., Milsom, M.D., Shaffer, J., Neuenfeldt, J., Shaaban, A.F., Margison, G.P., Fairbairn, L.J. and Chinnasamy, N. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI, Virol. J., 3: 14, 2006.

    Article  PubMed  Google Scholar 

  13. Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L. and Trono, D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo, Nat. Biotechnol., 15: 871–875, 1997.

    Article  PubMed  CAS  Google Scholar 

  14. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F.H., Verma, I.M. and Trono, D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector, Science, 272: 263–267, 1996.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dhanalakshmi Chinnasamy .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Chinnasamy, N., Shaffer, J., Chinnasamy, D. (2009). Production of Multicistronic HIV-1 Based Lentiviral Vectors. In: Hicks, B.W. (eds) Viral Applications of Green Fluorescent Protein. Methods in Molecular Biology™, vol 515. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-559-6_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-559-6_9

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-934115-87-9

  • Online ISBN: 978-1-59745-559-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics