Construction of a Safe and Efficient Retrovirus Packaging Cell Line

  • Dina Markowitz
  • Stephen Goff
  • Arthur Bank
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 34)


Ecotropic and amphotropic retrovirus packaging Cell lines have been constructed in which the helper virus genome have been separated onto two plasmids, and the ψ packaging signal and 3’ LTR have been removed. The gag and pol genes on one plasmid and the env gene on another plasmid were transfected into NIH 3T3 Cells. Packaging Cell lines produced by these transfected genes released titers of replication-defective retroviral vectors which were comparable to titers produced by packaging Cell lines containing the helper virus genome on one plasmid. There has been no evidence of recombination events between the ecotropic helper virus plasmids and the vector virus plasmid that would result in the generation of intact replication-competent virus. These results suggest that a packaging Cell line containing gag, pol and env on different plasmids is efficient and safe for use in retroviral gene gransfer.

Key Words

Retroviral gene transfer — packaging Cell line. 


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  1. 1.
    Cone, R.D., A. Webber-Benarous, D. Baorto, and R.C. Mulligan. 1987. Regulated expression of a complete human ß-globin gene encoded by a transmissible retrovirus vector. Mol. Cell. Biol. 7: 887–897.PubMedGoogle Scholar
  2. 2.
    Anderson, W.F. 1984. Prospects for human gene therapy. Science 226: 401–409.PubMedCrossRefGoogle Scholar
  3. 3.
    Neel, B.B., W.S. Hayward, H.L. Robinson, J. Fang, and S. M. Astrin. 1981. Avian leukosis virus-induced tumors have common proviral integration sites and synthesize discrete new RNAs: Oncogenesis by promoter insertion. Cell 23: 323–334.PubMedCrossRefGoogle Scholar
  4. 4.
    Varmus, H.E., N. Quintrell, and S. Ortiz. 1981. Retroviruses as mutagenss Insertion and exision of a nontransforming provirus alter expression of a resident transforming provirus. Cell 25: 23–36.PubMedCrossRefGoogle Scholar
  5. 5.
    Cone, R.D., and R.C. Mulligan. 1984. High efficiency gene transfer into mammalian Cells: Generation of helper-free retrovirus with broad mammalian host range. Proc. Natl. Acad. Sci. 81: 6349–6353.PubMedCrossRefGoogle Scholar
  6. 6.
    Mann, R., F.C. Mulligan, and D. Baltimore. 1983. Construction of a retrovirus packaging mutant and its use to produce helper- free defective retrovirus. Cell 33: 153–159.PubMedCrossRefGoogle Scholar
  7. 7.
    Miller, A.D., M.-F. Law, and I.M. Verma. 1985. Generation of helper-free amphotropic retroviruses that transduce a dominant- acting methotrexate-resistant dihydrofolate reductase gene. Mol. Cell Biol. 5: 431–437.PubMedGoogle Scholar
  8. 8.
    Sorge, J., D. Wright, V.D. Erdman, and A. Cutting. 1984. Amphotropic retrovirus system for human Cell gene transfer. Mol. Cell. Biol. 4: 1730–1737.PubMedGoogle Scholar
  9. 9.
    Watanabe, S., and H.M. Temin. 1983. Construction of a helper Cell line for Avian reticuloendotheliosis virus cloning factors. Mol. Cell. Biol. 3: 2241–2249.PubMedGoogle Scholar
  10. 10.
    Hock, R.A., and A.D. Miller. 1986. Retrovirus-mediated transfer and expression of drug-resistant genes in human haematopoietic progenitor Cells. Nature 320: 257–277.CrossRefGoogle Scholar
  11. 11.
    Miller, A.D., and C. Buttimore. 1986. Redesign of retrovirus packaging Cell lines to avoid recombination leading to helper virus production. Mol. Cell. Biol. 6: 2895–2902.PubMedGoogle Scholar
  12. 12.
    Mulligan, R.C., and P. Berg. 1980. Expression of a bacterial gene in mammalian Cells. Science 209: 175–183.CrossRefGoogle Scholar
  13. 13.
    Potter, H., L.W. Weir, and P. Leder. 1984. Enhancer-dependent expression of human K immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc. Natl. Acad. Sci. 81: 7161–7165.PubMedCrossRefGoogle Scholar
  14. 14.
    Goff, S.P., P. Traktman, and D. Baltimore. 1981. Isolation and properties of Molony murine leukemia virus mutants: Use of a rapid assay for release of virion reverse transcriptase. J. Virol. 38: 239–248.PubMedGoogle Scholar
  15. 15.
    Schwartzberg, P., J. Colicelli, and S. Goff. 1983. Deletion mutants of Molony murine leukemia virus which lack glycosylated gag protein are replication competent. J. Virol. 46: 538–546.PubMedGoogle Scholar
  16. 16.
    Keller, G., C. Paige, E. Gilboa, and E.F. Wagner. 1985. Expression of a foreign gene in myeloid and lymphoid Cells derived from multipotent haematopoietic precursors. Nature 318: 149–154.PubMedCrossRefGoogle Scholar
  17. 17.
    Murphy, A.J. 1987. Molecular techniques for the isolation of transcriptional transacting genes. Doctoral Thesis, Columbia University 133–139.Google Scholar

Copyright information

© Plenum Press, New York 1988

Authors and Affiliations

  • Dina Markowitz
    • 1
    • 2
    • 3
  • Stephen Goff
    • 1
    • 2
    • 3
  • Arthur Bank
    • 1
    • 2
    • 3
  1. 1.Department of Genetics and Development College of PhysiciansColumbia UniversityNew YorkUSA
  2. 2.Department of Biochemistry and Molecular Biophysics College of Physicians and SurgeonsColumbia UniversityNew YorkUSA
  3. 3.Department of Medicine College of Physicians and SurgeonsColumbia UniversityNew YorkUSA

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