Overview of the Retrovirus Transduction System

Constance Cepko1, Warren Pear2

1 Harvard Medical School, Boston, Massachusetts, 2 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 9.9
DOI:  10.1002/0471142727.mb0909s36
Online Posting Date:  May, 2001
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A retrovirus vector is an infectious virus used to introduce a nonviral gene into mitotic cells in vivo or in vitro. The efficient and precise integration machinery of naturally occurring retroviruses is utilized to produce either a single copy or a few copies of the viral genome stably integrated into a host chromosome. This unit presents an overview of the retrovirus life cycle and a description of vector designs and packaging cell lines.

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Table of Contents

  • SECTION III: Transduction of Genes Using Retrovirus Vectors
  • Retrovirus Life Cycle
  • Replication‐Incompetent Vectors
  • Replication‐Competent Vectors
  • Packaging Lines and Virus Production
  • Murine Retroviruses
  • Avian Retroviruses
  • Safety Issues
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Albritton, L.M., Tseng, L., Scadden, D., and Cunningham., J.M. 1989. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane–spanning protein and confers susceptibility to virus infection. Cell 57:659‐66.
   Bauer, T.R. Jr., Miller, A.D., and Hickstein, D.D. 1995. Improved transfer of the leukocyte integrin CD18 subunit into hematopoietic cell lines by using retroviral vectors having a gibbon ape leukemia virus envelope. Blood 86:2379‐2387.
   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.
   Boris‐Lawrie, K.A. and Temin, H.M. 1993. Recent advances in retrovirus vector technology. Curr. Opin. Genet. Dev. 3:102‐9.
   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. U.S.A. 90:8033‐8037.
   Cone, R.D. and Mulligan, R.C. 1984. High‐efficiency gene transfer into mammalian cells: Generation of helper‐free recombinant retrovirus with broad mammalian host range. Proc. Natl. Acad. Sci. U.S.A. 81:6349‐6353.
   Cosset, F.L., Legras, C., Chebloune, Y., Savatier, P., Thoraval, P., Thomas, J.L., Samarut, J., Nigon, V.M., and Verdier, G. 1990. A new avian leukosis virus–based packaging cell line that uses two separate transcomplementing helper genomes. J. Virol. 64:1070‐8.
   Cosset, F.L., Takeuchi, Y., Battini, J.L., Weiss, R.A., and Collins, M.K. 1995. High‐titer packaging cells producing recombinant retroviruses resistant to human serum. J. Virol. 69:7430‐7436.
   Danos, O. and Mulligan, R.C. 1988. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. U.S.A. 85:6460‐6464.
   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. U.S.A. 90:3539‐3543.
   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.
   Ghattas, I.R., Sanes, J.R., and Majors, J.E. 1991. The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol. Cell. Biol. 11:5848‐59.
   Gossen, M. and Bujard, H. 1992. Tight control of gene expression in mammalian cells by tetracycline‐responsive promoters. Proc. Natl. Acad. Sci. U.S.A. 89:5547‐5551.
   Han, X., Kasahara, N., and Kan, Y.W. 1995. Ligand‐directed retroviral targeting of human breast cancer cells. Proc. Natl. Acad. Sci. U.S.A. 92:9747‐9751.
   Hawley, R.G., Lieu, F.H., Fong, A.Z., and Hawley, T.S. 1994. Versatile retroviral vectors for potential use in gene therapy. Gene Ther. 1:136‐138.
   Hofmann, A., Nolan, G.P., and Blau, H.M. 1996. Rapid retroviral delivery of tetracycline‐inducible genes in a single autoregulatory cassette. Proc. Natl. Acad. Sci. U.S.A. 93:5185‐5190.
   Hughes, S.H., Greenhouse, J.J., Petropoulos, C.J., and Sutrave, P. 1987. Adaptor plasmids simplify the insertion of foreign DNA into helper‐independent retroviral vectors. J. Virol. 61:3004‐12.
   Kasahara, N., Dozy, A.M., and Kan, Y.W. 1994. Tissue‐specific targeting of retroviral vectors through ligand‐receptor interactions. Science 266:1373‐1376.
   Kavanaugh, M.P., Miller, D.G., Zhang, W., Law, W., Kozak, S.L., Kabat, D., and Miller, A.D. 1994. Cell‐surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium‐dependent phosphate symporters. Proc. Natl. Acad. Sci. U.S.A. 91:7071‐7075.
   Kim, J.W., Closs, E.I., Albritton, L.M., and Cunningham, J.M. 1991. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. Nature 352:725‐8.
   Landau, N.R. and Littman, D.R. 1992. Packaging system for rapid generation of murine leukemia virus vectors with variable tropism. J. Virol. 66:5110‐5113.
   Lodish, H., Baltimore, D., and Darnell, J. 1995. Molecular Cell Biology. W.H. Freeman, New York.
   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.
   Marin, M., Noel, D., Valsesia‐Wittman, S., Brockly, F., Etienne‐Julan, M., Russell, S., Cosset, F.K., and Piechaczyk, M. 1996. Targeted infection of human cells via major histocompatibility complex class I molecules by Moloney murine leukemia virus–derived viruses displaying single‐chain antibody fragment–envelope fusion proteins. J. Virol. 70:2957‐2962.
   Markowitz, D., Goff, S., and Bank, A. 1988a. A safe packaging line for gene transfer: Separating viral genes on two different plasmids. J. Virol. 62:1120‐1124.
   Markowitz, D., Goff, S., and Bank, A. 1988b. Construction and use of a safe and efficient amphotropic packaging cell line. Virology 167:400‐406.
   Miller, A.D. 1996. Retroviral vectors In Current Protocols in Human Genetics (N. Dracopoli, J. Haines, B.R. Korf, D.T. Moir, C.C. Morton, C.E. Seidman, J.G. Seidman, and D.R. Smith, eds.) pp. 12.5.1‐12.5.19. John Wiley & Sons, New York.
   Miller, A.D. and Buttimore, C. 1986. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell. Biol. 6:2895‐2902.
   Miller, A.D. and Chen, F. 1996. Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry. J. Virol. 70:5564‐5571.
   Miller, D.G. and Miller, A.D. 1994. A family of retroviruses that utilize related phosphate transporters for cell entry. J. Virol. 68:8270‐8276.
   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.
   Miller, A.D., Garcia, J.V., von Suhr, N., Lynch, C.M., Wilson, C., and Eiden, M.V. 1991. Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J. Virol. 65:2220‐4.
   Miller, D.G., Edwards, R.H., and Miller, A.D. 1994. Cloning of the cellular receptor for amphotropic murine retroviruses reveals homology to that for gibbon ape leukemia virus. Proc. Natl. Acad. Sci. U.S.A. 91:78‐82.
   Morgenstern, J.P. and Land, H. 1990. Advanced mammalian gene transfer: High‐titer retroviral vectors with multiple drug selection markers and a complementary helper‐free packaging cell line. Nucl. Acids Res. 18:3587‐3596.
   Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F.H., Verma, I.M., and Trono, D. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263‐267.
   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.
   Paulus, W., Baur, I., Boyce, F.M., Breakefield, X.O., and Reeves, S.A. 1996. Self‐contained, tetracycline‐regulated retroviral vector system for gene delivery to mammalian cells. J. Virol. 70:62‐67.
   Pear, W., Nolan, G., Scott, M., and Baltimore, D. 1993. Production of high‐titer helper‐free retroviruses by transient transfection. Proc. Natl. Acad. Sci. U.S.A. 90:8392‐8396.
   Pear, W.S., Scott, M.L., and Nolan, G.P. 1996. Generation of high‐titer, helper‐free retroviruses by transient transfection. In Methods in Molecular Biology: Methods in Gene Therapy (P. Robbins, ed.) in press. Humana Press, Totowa, N.J.
   Riviere, I. and Sadelain, M. 1996. Methods for the construction of retroviral vectors and the generation of high‐titer producers In Methods in Molecular Medicine: Gene Therapy Protocols (P. Robbins, ed.) in press. Humana Press, Totowa, N.J.
   Roe, T., Reynolds, T.C., Yu, G., and Brown, P.O. 1993. Integration of murine leukemia virus DNA depends on mitosis. EMBO J. 12:2099‐108.
   Shockett, P.E. and Schatz, D.G. 1996. Diverse strategies for tetracycline‐regulated inducible gene expression. Proc. Natl. Acad. Sci. U.S.A. 93:5173‐5176.
   Somia, N.V., Zoppe, M., and Verma, I.M. 1995. Generation of targeted retroviral vectors by using single‐chain variable fragment: An approach to in vivo gene delivery. Proc. Natl. Acad. Sci. U.S.A. 92:7570‐7574.
   Soneoka, Y., Cannon, P.M., Ramsdale, E.E., Griffiths, J.C., Romano, G., Kingsman, S.M., and Kingsman, A.J. 1995. A transient three‐plasmid expression system for the production of high titer retroviral vectors. Nucl. Acids Res. 23:628‐633.
   Stoker, A.W. and Bissell, M.J. 1988. Development of avian sarcoma and leukosis virus–based vector‐packaging cell lines. J. Virol. 62:1008‐1015.
   Takahara, Y., Hamada, K., and Housman, D.E. 1992. A new retrovirus packaging cell for gene transfer constructed from amplified long terminal repeat–free chimeric proviral genes. J. Virol. 66:3725‐32.
   Takeuchi, Y., Vile, R.G., Simpson, G., O'Hara, B., Collins, M.K., and Weiss, R.A. 1992. Feline leukemia virus subgroup B uses the same cell surface receptor as gibbon ape leukemia virus. J. Virol. 66:1219‐22.
   van Zeijl, M., Johann, S.V., Closs, E., Cunningham, J., Eddy, R., Shows, T.B., and O'Hara, B. 1994. A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family. Proc. Natl. Acad. Sci. U.S.A. 91:1168‐72.
   Wang, H., Kavanaugh, M.P., North, R.A., and Kabat, D. 1991. Cell‐surface receptor for ecotropic murine retroviruses is a basic amino‐acid transporter. Nature 352:729‐31.
   Yang, Y., Vanin, E.F., Whitt, M.A., Fornerod, M., Zwart, R., Schneiderman, R.D., Grosveld, G., and Nienhuis, A.W. 1995. Inducible, high‐level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein. Hum. Gene Ther. 6:1203‐1213.
   Yee, J.K., Friedmann, T., and Burns, J.C. 1994. Generation of high‐titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol. 43:99‐112.
   Yu, S.F., von Ruden, T., Kantoff, P.W., Garber, C., Seiberg, M., Ruther, U., Anderson, W.F., Wagner, E.F., and Gilboa, E. 1986. Self‐inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 83:3194‐3198.
   Zitvogel, L., Tahara, H., Cai, Q., Storkus, W.J., Muller, G., Wolf, S.F., Gately, M., Robbins, P.D., and Lotze, M.T. 1994. Construction and characterization of retroviral vectors expressing biologically active human interleukin‐12. Hum. Gene Ther. 5:1493‐1506.
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