Abstract
Over the past decade, lentiviral vectors have emerged as powerful tools for transgene delivery. The use of lentiviral vectors has become commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis are rapidly emerging. Also, lentiviral vectors are at present being explored in the context of human clinical trials. Here we describe improved protocols to generate highly concentrated lentiviral vector pseudotypes involving different envelope glycoproteins. In this protocol, vector stocks are prepared by transient transfection using standard cell culture media or serum-free media. Such stocks are then concentrated by ultracentrifugation and/or ion exchange chromatography, or by precipitation using polyethylene glycol 6000, resulting in vector titers of up to 1010 transducing units per milliliter and above. We also provide reliable real-time PCR protocols to titrate lentiviral vectors based on proviral DNA copies present in genomic DNA extracted from transduced cells or on vector RNA. These production/concentration methods result in high-titer vector preparations that show reduced toxicity compared with lentiviral vectors produced using standard protocols involving ultracentrifugation-based methods. The vector production and titration protocol described here can be completed within 8 d.
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References
Akkina, R.K. et al. High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G. J. Virol. 70, 2581–2585 (1996).
Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).
Reiser, J. et al. Transduction of nondividing cells using pseudotyped defective high- titer HIV type 1 particles. Proc. Natl. Acad. Sci. USA 93, 15266–15271 (1996).
Reiser, J., Lai, Z., Zhang, X.Y. & Brady, R.O. Development of multigene and regulated lentivirus vectors. J. Virol. 74, 10589–10599 (2000).
Cronin, J., Zhang, X.Y. & Reiser, J. Altering the tropism of lentiviral vectors through pseudotyping. Curr. Gene. Ther. 5, 387–398 (2005).
Reiser, J. Production and concentration of pseudotyped HIV-1-based gene transfer vectors. Gene Ther. 7, 910–913 (2000).
Sena-Esteves, M., Tebbets, J.C., Steffens, S., Crombleholme, T. & Flake, A.W. Optimized large-scale production of high titer lentivirus vector pseudotypes. J. Virol. Methods 122, 131–139 (2004).
Chen, S.T., Iida, A., Guo, L., Friedmann, T. & Yee, J.K. Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system. Proc. Natl. Acad. Sci. USA 93, 10057–10062 (1996).
Zhang, X.Y., La Russa, V.F. & Reiser, J. Transduction of bone-marrow-derived mesenchymal stem cells by using lentivirus vectors pseudotyped with modified RD114 envelope glycoproteins. J. Virol. 78, 1219–1229 (2004).
Ricks, D.M., Kutner, R., Zhang, X.Y., Welsh, D.A. & Reiser, J. Optimized lentiviral transduction of mouse bone marrow-derived mesenchymal stem cells. Stem. Cells Dev. 17, 441–450 (2008).
Scherr, M., Battmer, K., Blomer, U., Ganser, A. & Grez, M. Quantitative determination of lentiviral vector particle numbers by real-time PCR. Biotechniques 31, 520–522, 524, passim (2001).
Logan, A.C. et al. Factors influencing the titer and infectivity of lentiviral vectors. Hum. Gene. Ther. 15, 976–988 (2004).
Radcliffe, P.A. et al. Analysis of factor VIII mediated suppression of lentiviral vector titres. Gene Ther. 15, 289–297 (2008).
Zhang, X.Y. et al. Lentiviral vectors for sustained transgene expression in human bone marrow-derived stromal cells. Mol. Ther. 5, 555–565 (2002).
Sastry, L., Johnson, T., Hobson, M.J., Smucker, B. & Cornetta, K. Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Gene Ther. 9, 1155–1162 (2002).
Pluta, K., Luce, M.J., Bao, L., Agha-Mohammadi, S. & Reiser, J. Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters. J. Gene. Med. 7, 803–817 (2005).
Pluta, K. et al. Lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study. BMC Biotechnol. 7, 41 (2007).
Kuroda, H., Kutner, R.H., Bazan, N.G. & Reiser, J. A comparative analysis of constitutive and cell-specific promoters in the adult mouse hippocampus using lentivirus vector-mediated gene transfer. J. Gene. Med. 10, 1163–1175 (2008).
Baekelandt, V., Eggermont, K., Michiels, M., Nuttin, B. & Debyser, Z. Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain. Gene Ther. 10, 1933–1940 (2003).
Marino, M.P., Luce, M.J. & Reiser, J. Small- to large-scale production of lentivirus vectors. In Lentivirus Gene Engineering Protocols, Vol. 229 (ed. M. Federico) 43–55 (Humana Press, Totowa, NJ, 2003).
Mochizuki, H., Schwartz, J.P., Tanaka, K., Brady, R.O. & Reiser, J. High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells. J. Virol. 72, 8873–8883 (1998).
Follenzi, A. & Naldini, L. Generation of HIV-1 derived lentiviral vectors. Methods Enzymol. 346, 454–465 (2002).
Teng, M.N., Borrow, P., Oldstone, M.B. & de la Torre, J.C. A single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with the ability to cause growth hormone deficiency syndrome. J. Virol. 70, 8438–8443 (1996).
Cornu, T.I. & de la Torre, J.C. Characterization of the arenavirus RING finger Z protein regions required for Z-mediated inhibition of viral RNA synthesis. J. Virol. 76, 6678–6688 (2002).
Amendola, M., Venneri, M.A., Biffi, A., Vigna, E. & Naldini, L. Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat. Biotechnol. 23, 108–116 (2005).
Strang, B.L., Ikeda, Y., Cosset, F.L., Collins, M.K. & Takeuchi, Y. Characterization of HIV-1 vectors with gammaretrovirus envelope glycoproteins produced from stable packaging cells. Gene Ther. 11, 591–598 (2004).
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This work was supported by NIH grant NS044832.
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Kutner, R., Zhang, XY. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 4, 495–505 (2009). https://doi.org/10.1038/nprot.2009.22
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DOI: https://doi.org/10.1038/nprot.2009.22
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