Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Aug;70(8):5701–5705. doi: 10.1128/jvi.70.8.5701-5705.1996

The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.

R K Naviaux 1, E Costanzi 1, M Haas 1, I M Verma 1
PMCID: PMC190538  PMID: 8764092

Abstract

We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 X 10(6) to 5 X 10(6) within 48 h. These vectors contain the immediate early region of the human cytomegalovirus enhancer-promoter fused to the Moloney murine leukemia virus long terminal repeat at the TATA box in the 5' U3 region, yielding the pCL promoter. By selecting vectors designed to express genes from one of four promoters (dihydrofolate reductase, Rous sarcoma virus, long terminal repeat, or cytomegalovirus), the pCL system permits the investigator to control the level of gene expression in target cells over a 100-fold range, while maintaining uniformly high titers of virus from transiently transfected producer cells. The pCL packaging plasmids lack a packaging signal (delta-psi) and include an added safety modification that renders them self-inactivating through the deletion of the 3' U3 enhancer. Ecotropic, amphotropic (4070A), and amphotropic-mink cell focus-forming hybrid (10A1) envelope constructions have been prepared and tested, permitting flexible selection of vector pseudotype in accordance with experimental needs. Vector supernatants are free of helper virus and are of sufficiently high titer within 2 days of transient transfection in 293 cells to permit infection of more than 50% of randomly cycling target cells in culture. We demonstrated the efficacy of these vectors by using them to transfer three potent cell cycle control genes (the p16(INK4A), p53, and Rb1 genes) into human glioblastoma cells.

Full Text

The Full Text of this article is available as a PDF (216.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burns J. C., Friedmann T., Driever W., Burrascano M., Yee J. K. 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 U S A. 1993 Sep 1;90(17):8033–8037. doi: 10.1073/pnas.90.17.8033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cornetta K., Nguyen N., Morgan R. A., Muenchau D. D., Hartley J. W., Blaese R. M., Anderson W. F. Infection of human cells with murine amphotropic replication-competent retroviruses. Hum Gene Ther. 1993 Oct;4(5):579–588. doi: 10.1089/hum.1993.4.5-579. [DOI] [PubMed] [Google Scholar]
  4. Danos O., Mulligan R. C. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6460–6464. doi: 10.1073/pnas.85.17.6460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Finer M. H., Dull T. J., Qin L., Farson D., Roberts M. R. kat: a high-efficiency retroviral transduction system for primary human T lymphocytes. Blood. 1994 Jan 1;83(1):43–50. [PubMed] [Google Scholar]
  6. Goff S., Traktman P., Baltimore D. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J Virol. 1981 Apr;38(1):239–248. doi: 10.1128/jvi.38.1.239-248.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  8. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  9. Gudkov A. V., Kazarov A. R., Thimmapaya R., Axenovich S. A., Mazo I. A., Roninson I. B. Cloning mammalian genes by expression selection of genetic suppressor elements: association of kinesin with drug resistance and cell immortalization. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3744–3748. doi: 10.1073/pnas.91.9.3744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hartley J. W., Rowe W. P. Clonal cells lines from a feral mouse embryo which lack host-range restrictions for murine leukemia viruses. Virology. 1975 May;65(1):128–134. doi: 10.1016/0042-6822(75)90013-6. [DOI] [PubMed] [Google Scholar]
  11. Landau N. R., Littman D. R. Packaging system for rapid production of murine leukemia virus vectors with variable tropism. J Virol. 1992 Aug;66(8):5110–5113. doi: 10.1128/jvi.66.8.5110-5113.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  13. Markowitz D., Goff S., Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988 Apr;62(4):1120–1124. doi: 10.1128/jvi.62.4.1120-1124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Markowitz D., Goff S., Bank A. Construction and use of a safe and efficient amphotropic packaging cell line. Virology. 1988 Dec;167(2):400–406. [PubMed] [Google Scholar]
  15. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Miller A. D., Law M. F., Verma I. M. Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene. Mol Cell Biol. 1985 Mar;5(3):431–437. doi: 10.1128/mcb.5.3.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Miller A. D., Miller D. G., Garcia J. V., Lynch C. M. Use of retroviral vectors for gene transfer and expression. Methods Enzymol. 1993;217:581–599. doi: 10.1016/0076-6879(93)17090-r. [DOI] [PubMed] [Google Scholar]
  18. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  19. Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. doi: 10.1093/nar/18.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Okamoto A., Demetrick D. J., Spillare E. A., Hagiwara K., Hussain S. P., Bennett W. P., Forrester K., Gerwin B., Serrano M., Beach D. H. Mutations and altered expression of p16INK4 in human cancer. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11045–11049. doi: 10.1073/pnas.91.23.11045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ott D., Friedrich R., Rein A. Sequence analysis of amphotropic and 10A1 murine leukemia viruses: close relationship to mink cell focus-inducing viruses. J Virol. 1990 Feb;64(2):757–766. doi: 10.1128/jvi.64.2.757-766.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pear W. S., Nolan G. P., Scott M. L., Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392–8396. doi: 10.1073/pnas.90.18.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rowe W. P., Pugh W. E., Hartley J. W. Plaque assay techniques for murine leukemia viruses. Virology. 1970 Dec;42(4):1136–1139. doi: 10.1016/0042-6822(70)90362-4. [DOI] [PubMed] [Google Scholar]
  24. Sanes J. R., Rubenstein J. L., Nicolas J. F. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986 Dec 1;5(12):3133–3142. doi: 10.1002/j.1460-2075.1986.tb04620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Soneoka Y., Cannon P. M., Ramsdale E. E., Griffiths J. C., Romano G., Kingsman S. M., Kingsman A. J. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 1995 Feb 25;23(4):628–633. doi: 10.1093/nar/23.4.628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wong B. Y., Chen H., Chung S. W., Wong P. M. High-efficiency identification of genes by functional analysis from a retroviral cDNA expression library. J Virol. 1994 Sep;68(9):5523–5531. doi: 10.1128/jvi.68.9.5523-5531.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yu S. F., von Rüden T., Kantoff P. W., Garber C., Seiberg M., Rüther U., Anderson W. F., Wagner E. F., Gilboa E. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3194–3198. doi: 10.1073/pnas.83.10.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES