Abstract
Multiplasmid transient transfection is the most widely used technique for the generation of lentiviral vectors. However, traditional transient transfection protocols using 293 T adherent cells and calcium phosphate/DNA co-precipitation followed by ultracentrifugation are tedious, time-consuming, and difficult to scale up. This chapter describes a streamlined protocol for the fast mass production of lentiviral vectors and their purification by affinity chromatography. Lentiviral particles are generated by transient transfection of suspension growing HEK 293 cells in serum-free medium using polyethylenimine (PEI) as transfection reagent. Lentiviral vector production is carried out in Erlenmeyer flasks agitated on orbital shakers requiring minimum supplementary laboratory equipment. Alternatively, the method can be easily scaled up to generate larger volumes of vector stocks in bioreactors. Heparin affinity chromatography allows for selective concentration and purification of lentiviral particles in a singlestep directly from vector supernatants. The method is suitable for the production and purification of different vector pseudotypes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Naldini, L., Blomer, U., Gallay, P., Ory, P., Mulligan, R., Gage, F.H., et al. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263-7.
Kafri, T., van Praag, H., Ouyang, L., Gage, F.H., and Verma, I.M. (1999). A packaging cell line for lentivirus vectors. J Virol 73, 576-84.
Lesch, H.P., Turpeinen, S., Niskanen, E.A., Mähönen, A.J., Airenne, K.J., and Ylä-Herttuala, S. (2008). Generation of lentivirus vectors using recombinant baculoviruses. Gene Ther 15, 1280-6.
Bartz, S.R., Rogel, M.E., and Emerman, M. (1996). Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control. J Virol 70, 2324-31.
Konvalinka, J., Litterst, M.A., Welker, R., Kottler, H., Rippmann, F., Heuser, A.M., et al. (1995). An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity. J Virol 69, 7180-6.
Li, C.J., Friedman, D.J., Wang, C., Metelev, V., and Pardee, A.B. (1995). Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein. Science 268, 429-31.
Miyazaki, Y., Takamatsu, T., Nosaka, T., Fujita, S., Martin, T.E., and Hatanaka, M. (1995). The cytotoxicity of human immunodeficiency virus type 1 Rev: implications for its interaction with the nucleolar protein B23. Exp Cell Res 219, 93-101.
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 U S A 90, 8033-7.
Broussau, S., Jabbour, N., Lachapelle, G., Durocher, Y., Tom, R., Transfiguracion, J., et al. (2008). Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture. Mol Ther 16, 500-7.
Sena-Esteves, M., Tebbets, J.C., Steffens, S., Crombleholme, T., and Flake, A.W. (2004). Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods 122, 131-9.
Follenzi, A. and Naldini, L. (2002). HIV-based vectors. Preparation and use. Methods Mol Med 69, 259-74.
Tiscornia, G., Singer, O., and Verma, I.M. (2006). Production and purification of lentiviral vectors. Nat Protoc 1, 241-5.
Salmon, P. and Trono, D. (2007). Production and titration of lentiviral vectors. Curr Protoc Hum Genet Chapter 12, Unit 12 10.
Tonini, T., Claudio, P.P., Giordano, A., and Romano, G. (2004). Transient production of retroviral- and lentiviral-based vectors for the transduction of Mammalian cells. Methods Mol Biol 285, 141-8.
Chang, L.J. and Zaiss, A.K. (2002). Lentiviral vectors. Preparation and use. Methods Mol Med 69, 303-18.
Segura, M.M., Garnier, A., Durocher, Y., Coelho, H., and Kamen, A. (2007). Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification. Biotechnol Bioeng 98, 789-99.
Durocher, Y., Perret, S., and Kamen, A. (2002). High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30, E9.
Dull, T., Zufferey, R., Kelly, M., Mandel, R.J., Nguyen, M., Trono, D., et al. (1998). A third-generation lentivirus vector with a conditional packaging system. J Virol 72, 8463-71.
Segura, M.M., Kamen, A., and Garnier, A. (2006). Downstream processing of oncoretroviral and lentiviral gene therapy vectors. Biotechnol Adv 24, 321-37.
Segura, M.M., Kamen, A., and Garnier, A. (2008). Purification of retrovirus particles using heparin affinity chromatography. Methods Mol Biol 434, 1-11.
Segura, M.M., Kamen, A., Lavoie, M.C., and Garnier, A. (2007). Exploiting heparin-binding properties of MoMLV-based retroviral vectors for affinity chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 846, 124-31.
Segura, M.M., Kamen, A., Trudel, P., and Garnier, A. (2005). A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography. Biotechnol Bioeng 90, 391-404.
Kobinger, G.P., Weiner, D.J., Yu, Q.C., and Wilson, J.M. (2001). Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nat Biotechnol 19, 225-30.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Segura, M.M., Garnier, A., Durocher, Y., Ansorge, S., Kamen, A. (2010). New Protocol for Lentiviral Vector Mass Production. In: Federico, M. (eds) Lentivirus Gene Engineering Protocols. Methods in Molecular Biology, vol 614. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-533-0_2
Download citation
DOI: https://doi.org/10.1007/978-1-60761-533-0_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-532-3
Online ISBN: 978-1-60761-533-0
eBook Packages: Springer Protocols