Murine and HIV-Based Retroviral Vectors for In Vitro and In Vivo Gene Transfer

  • Ronald W. Alfa
  • Armin Blesch
Part of the Methods in Molecular Medicine book series (MIMM, volume 129)


The success of experimental gene therapy is dependent on the ability to safely and efficiently introduce transgenes into the target cell or tissue. Retroviral-based vectors, notably those derived from Moloney murine leukemia virus (MLV) and lentiviral vectors derived from HIV, have proven to be valuable gene transfer vehicles as a result of their ease of production and their ability to mediate long-term transgene expression. One of the most widely used methods for viral vector production is based on the transient transfection of viral vector plasmid DNA into a producer cell line. Here, we describe protocols to produce and standardize high quality MLV-based retroviral and HIV-based lentiviral vectors for ex vivo and in vivo gene delivery.

Key Words

Gene therapy lentivirus retrovirus HIV virus producer cells 


  1. 1.
    Pages, J. C. and Bru, T. (2004) Toolbox for retrovectorologists. J. Gene Med. 6(Suppl 1), S67–S82.CrossRefPubMedGoogle Scholar
  2. 2.
    Weiss, R. (1998) Viral RNA-dependent DNA polymerase RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Rev. Med. Virol. 8, 3–11.CrossRefPubMedGoogle Scholar
  3. 3.
    Southgate, C., Zapp, M. L., and Green, M. R. (1990) Activation of transcription by HIV-1 Tat protein tethered to nascent RNA through another protein. Nature 345, 640–642.CrossRefPubMedGoogle Scholar
  4. 4.
    Kim, V. N., Mitrophanous, K., Kingsman, S. M., and Kingsman, A. J. (1998) Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J. Virol. 72, 811–816.PubMedGoogle Scholar
  5. 5.
    Farnet, C. M. and Bushman, F. D. (1997) HIV-1 cDNA integration: requirement of HMG I(Y) protein for function of preintegration complexes in vitro. Cell 88, 483–492.CrossRefPubMedGoogle Scholar
  6. 6.
    Bushman, F. D. (1999) Host proteins in retroviral cDNA integration. Adv. Virus Res. 52, 301–317.CrossRefPubMedGoogle Scholar
  7. 7.
    Bukrinsky, M. I., Haggerty, S., Dempsey, M. P., et al. (1993) A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365, 666–669.CrossRefPubMedGoogle Scholar
  8. 8.
    Gallay, P., Swingler, S., Aiken, C., and Trono D. (1995) HIV-1 infection of nondividing cells: C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator. Cell 80, 379–388.CrossRefPubMedGoogle Scholar
  9. 9.
    Gallay, P., Swingler, S., Song, J., Bushman, F., and Trono, D. (1995) HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell 83, 569–576.CrossRefPubMedGoogle Scholar
  10. 10.
    Roe, T., Reynolds, T. C., Yu, G., and Brown, P. O. (1993) Integration of murine leukemia virus DNA depends on mitosis. EMBO J. 12, 2099–2108.PubMedGoogle Scholar
  11. 11.
    Miller, D. G., Adam, M. A., and Miller, A.D. (1990) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell Biol. 10, 4239–4242.PubMedGoogle Scholar
  12. 12.
    Linial, M. L. and Miller, A. D. (1990) Retroviral RNA packaging: sequence requirements and implications. Curr. Top Microbiol. Immunol. 157, 125–152.PubMedGoogle Scholar
  13. 13.
    Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D. (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA 90, 8392–8396.CrossRefPubMedGoogle Scholar
  14. 14.
    Blesch, A. (2004) Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer. Methods 33, 164–172.CrossRefPubMedGoogle Scholar
  15. 15.
    Palmer, T. D. and Gage, F. H. (1996) Delivery of gene products to the central nervous system by grafting retrovirally engineered neuronal and nonneuronal cells, in Protocols for Gene Transfer in Neuroscience: Towards Gene Therapy of Neurological Disorders, (Lowenstein, P. R. and Enquist, L.W., eds.). John Wiley and Sons Ltd, New York pp. 235–262.Google Scholar
  16. 16.
    Miller, A. D., Trauber, D. R., and Buttimore, C. (1986) Factors involved in production of helper virus-free retrovirus vectors. Somat. Cell Mol. Genet. 12, 175–183.CrossRefPubMedGoogle Scholar
  17. 17.
    Miller, A. D., Miller, D. G., Garcia, J. V., and Lynch, C. M. (1993) Use of retroviral vectors for gene transfer and expression. Methods Enzymol. 217, 581–599.CrossRefPubMedGoogle Scholar
  18. 18.
    Soneoka, Y., Cannon, P. M., Ramsdale, E. E., et al. (1995) A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23, 628–633.CrossRefPubMedGoogle Scholar
  19. 19.
    Palu, G., Parolin, C., Takeuchi, Y., and Pizzato, M. (2000) Progress with retroviral gene vectors. Rev. Med. Virol. 10, 185–202.CrossRefPubMedGoogle Scholar
  20. 20.
    Naviaux, R. K., Costanzi, E., Haas, M., and Verma, I. M. (1996) The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 70, 5701–5705.PubMedGoogle Scholar
  21. 21.
    Yee, J. K., Moores, J. C., Jolly, D. J., et al. (1987) Gene expression from transcriptionally disabled retroviral vectors. Proc. Natl. Acad. Sci. USA 84, 5197–5201.CrossRefPubMedGoogle Scholar
  22. 22.
    Miyoshi, H., Blomer, U., Takahashi, M., Gage, F. H., and Verma, I. M. (1998) Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150–8157.PubMedGoogle Scholar
  23. 23.
    Varmus, H. (1988) Retroviruses. Science 240, 1427–1435.CrossRefPubMedGoogle Scholar
  24. 24.
    Miller, A. D. (1996) Cell-surface receptors for retroviruses and implications for gene transfer. Proc. Natl. Acad. Sci. USA 93, 11,407–11,413.CrossRefPubMedGoogle Scholar
  25. 25.
    Chen, S. T., Iida, A., Guo, L., Friedmann, T., and Yee, J. K. (1996) Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system. Proc. Natl. Acad. Sci. USA 93, 10,057–10,062.CrossRefPubMedGoogle Scholar
  26. 26.
    Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J. K. (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. USA 90, 8033–8037.CrossRefPubMedGoogle Scholar
  27. 27.
    Yee, J. K., Miyanohara, A., LaPorte, P., Bouic, K., Burns, J. C., and Friedmann, T. (1994) A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes. Proc. Natl. Acad. Sci. USA 91, 9564–9568.CrossRefPubMedGoogle Scholar
  28. 28.
    Naldini, L., Blomer, U., Gage, F. H., Trono, D., and Verma, I. M. (1996) Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. USA 93, 11,382–11,388.CrossRefPubMedGoogle Scholar
  29. 29.
    Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., and Trono, D. (1997) Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15, 871–875.CrossRefPubMedGoogle Scholar
  30. 30.
    Dull, T., Zufferey, R., Kelly, M., et al. (1998) A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471.PubMedGoogle Scholar
  31. 31.
    Zufferey, R., Dull, T., Mandel, R. J., et al. (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873–9880.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Ronald W. Alfa
    • 1
  • Armin Blesch
    • 1
  1. 1.Department of NeurosciencesUniversity of CaliforniaSan Diego, La Jolla

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