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Production and purification of lentiviral vectors

Abstract

Lentiviral vectors offer unique versatility and robustness as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the transgene both in vitro and in vivo. This protocol describes how lentiviral vectors can be produced, purified and titrated. High titer suspensions can be routinely prepared with relative ease: a low-titer (106 viral particles/ml) unpurified preparation can be obtained 3 d after transfecting cells with lentiviral vector and packaging plasmids; a high-titer (109 viral particles/ml) purified preparation requires 2 more days.

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Figure 1: Schematic representation of the third generation lentiviral system.

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References

  1. Tiscornia, G., Singer, O. & Verma, I.M. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat. Protocols (2006) doi 10.1038/nprot.2006.36.

  2. Trono, D. Lentiviral Vectors (Berlin-Heidelberg, Springer-Verlag, 2002).

    Book  Google Scholar 

  3. Delenda, C. Lentiviral vectors: optimization of packaging, transduction and gene expression. J. Gene Med. 6, S125–S138 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

    Article  CAS  PubMed  Google Scholar 

  6. Brenner, S. & Malech, H.L. Current developments in the design of onco-retrovirus and lentivirus vector systems for hematopoietic cell gene therapy. Biochim. Biophys. Acta 1640, 1–24 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Rubinson, D.A. et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Tiscornia, G., Singer, O., Ikawa, M. & Verma, I.M. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc. Natl. Acad. Sci. USA 100, 1844–1848 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Miyoshi, H., Blomer, U., Takahashi, M., Gage, F.H. & Verma, I.M. Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150–8157 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Zufferey, R. et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873–9880 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Stevenson, M. Molecular biology of lentivirus-mediated gene transfer. Curr. Top. Microbiol. Immunol. 261, 1–30 (2002).

    CAS  PubMed  Google Scholar 

  12. Cronin, J., Zhang, X.Y. & Reiser, J. Altering the tropism of lentiviral vectors through pseudotyping. Curr. Gene Ther. 5, 387–398 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sandrin, V., Russell, S.J. & Cosset, F.L. Targeting retroviral and lentiviral vectors. Curr. Top. Microbiol. Immunol. 281, 137–178 (2003).

    CAS  PubMed  Google Scholar 

  14. Zufferey, R. Production of lentiviral vectors. Curr. Top. Microbiol. Immunol. 261, 107–121 (2002).

    CAS  PubMed  Google Scholar 

  15. Sinn, P.L., Sauter, S.L. & McCray, P.B. Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors—design, biosafety, and production. Gene Ther. 12, 1089–1098 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Saenz, D.T. & Poeschla, E.M. FIV: from lentivirus to lentivector. J. Gene Med. 6, S95–S104 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Logan, A.C., Haas, D.L., Kafri, T. & Kohn, D.B. Integrated self-inactivating lentiviral vectors produce full-length genomic transcripts competent for encapsidation and integration. J. Virol. 78, 8421–8436 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Pfeifer, A., Ikawa, M., Dayn, Y. & Verma, I.M. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc. Natl. Acad. Sci. USA 99, 2140–2145 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lois, C., Hong, E.J., Pease, S., Brown, E.J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Verhoeyen, E. & Cosset, F.L. Surface-engineering of lentiviral vectors. J. Gene Med. 6, S83–S94 (2004).

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Inder M Verma.

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Tiscornia, G., Singer, O. & Verma, I. Production and purification of lentiviral vectors. Nat Protoc 1, 241–245 (2006). https://doi.org/10.1038/nprot.2006.37

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