Skip to main content

Generation of High-Titer, Helper-Free Retroviruses by Transient Transfection

  • Protocol
Book cover Gene Therapy Protocols

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 7))

Abstract

Retroviral gene transfer is presently one of the most powerful techniques for introducing stably heritable genetic material into mammalian cells (reviewed in ref. 1). One serious drawback of this technique, however, has been the difficulty in readily producing high-titer recombinant retroviruses. For many applications, such as infecting rare target cells or the majority of cells in tissue culture, the recombinant virus titer must be at least 106 infectious units/mL. Although one can usually obtain high-titer mixtures of recombinant and replication-competent retroviruses in a relatively short time, many applications such as cell marking studies or studying genes in vivo demand freedom from replication-competent virus.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mulligan, R. C. (1993) The basic science of gene therapy. Science 260, 926–932.

    Article  CAS  Google Scholar 

  2. Mann, R., Mulligan, R. C, and Baltimore, D. (1983) Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33, 153–159.

    Article  CAS  Google Scholar 

  3. Watanabe, S. and Temin, H. M. (1983) Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. Mol Cell. Biol. 3, 2241–2249.

    CAS  Google Scholar 

  4. Miller, A. D. (1990) Retrovirus packaging cells. Human Gene Ther. 1, 5–14.

    Article  CAS  Google Scholar 

  5. Danos, O. (1991) Construction of retroviral packaging cell lines, in Methods in Molecular Biology, vol. 8 (Collins, M. ed.), Humana, Clifton, NJ, pp. 17–26.

    Google Scholar 

  6. Landau, N. R. and Littman, D. R. (1992) Packaging system for rapid generation of murine leukemia virus vectors with variable tropism. J. Viral 66, 5110–5113.

    Article  CAS  Google Scholar 

  7. Pear, W., Nolan, G., Scott, M., and Baltimore, D. (1993) Production of high-titer helperfree retroviruses by transient transfection. Proc. Natl. Acad. Sci USA 90, 8392–8396.

    Article  CAS  Google Scholar 

  8. Finer, M. H., Dull, T. J., Qin, L., Farson, D., and Roberts, M. R. (1994) Kat: a high-efficiency retroviral transduction system for primary human T lymphocytes. Blood 83, 43–50.

    Article  CAS  Google Scholar 

  9. Scadden, D. T., Fuller, B., and Cunningham, J. M. (1990) Human cells Infected with retrovirus vectors acquire an endogenous murine provirus. J Virol 64, 424–427.

    Article  CAS  Google Scholar 

  10. Graham, F. L., Smiley, J., Russell, W. C., and Naish, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen Virol. 36, 59–72.

    Article  CAS  Google Scholar 

  11. DuBridge, R. B., Tang, P., Hsia, H C., Phaik-Mooi, L, Miller, J H, and Calos, M. P. (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system Mol Cell. Biol. 7, 379–387.

    CAS  Google Scholar 

  12. Danos, O. and Mulligan, R. C. (1988) Safe and efficient generation of recombi-nant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl Acad Sci. USA 85, 6460–6464.

    Article  CAS  Google Scholar 

  13. Bender, M A., Palmer, T D, Gelinas, R. E, and Miller, A. D (1987) Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 61, 1639–1646.

    Article  CAS  Google Scholar 

  14. Daley, G. and Baltimore, D. (1988) Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210 bcr / abl protein. Proc. Natl Acad Sci USA 85, 9312–9316.

    Article  CAS  Google Scholar 

  15. Morgenstern, J. P. and Land, H. (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18, 3587–3596.

    Article  CAS  Google Scholar 

  16. Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K, Jackson, V., Hamada, H., Pardoll, D., and Mulligan, R C. (1993) Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl. Acad. Sci USA 90, 3539–3543.

    Article  CAS  Google Scholar 

  17. Shoemaker, C, Hoffmann, J., Goff, S. P, and Baltimore, D. (1981) Intramolecular integration within moloney murine leukemia virus DNA. J Viral 40, 164–172.

    Article  CAS  Google Scholar 

  18. Bernard, H. U., Krammer, G., and Rowekamp, W. G. (1985) Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture. Exp. Cell Res 158, 237–243.

    Article  CAS  Google Scholar 

  19. Goff, S., Traktman, P., and Baltimore, D. (1981) Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase .J Virol. 38, 239–248.

    Article  CAS  Google Scholar 

  20. Jasin, M. and Berg, P. (1988) Homologous integration in mammalian cells with-out target gene selection. Genes Dev 2, 1353–1363.

    Article  CAS  Google Scholar 

  21. Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J. K. (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to a very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl. Acad. Sci. USA 90, 8033–8037.

    Article  CAS  Google Scholar 

  22. Mulligan, R. C. and Berg, P. (1981) Selection for animal cells that express the escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc. Natl. Acad. Sci. USA 78, 2072–2076.

    Article  CAS  Google Scholar 

  23. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Strubl, K. (1989) Current Protocols in Molecular Biology, Wiley, New York.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1997 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Pear, W.S., Scott, M.L., Nolan, G.P. (1997). Generation of High-Titer, Helper-Free Retroviruses by Transient Transfection. In: Robbins, P.D. (eds) Gene Therapy Protocols. Methods in Molecular Medicine, vol 7. Humana, Totowa, NJ. https://doi.org/10.1385/0-89603-484-4:41

Download citation

  • DOI: https://doi.org/10.1385/0-89603-484-4:41

  • Publisher Name: Humana, Totowa, NJ

  • Print ISBN: 978-0-89603-484-6

  • Online ISBN: 978-1-59259-591-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics