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Lentiviral Transgenesis

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 530))

Abstract

Lentiviral vectors efficiently transfer genes into a broad spectrum of cells and tissues, including non-dividing cells and stem cells. Lentiviruses integrate their viral genome into the host chromosome, which is the basis for virus latency as well as stable transgene expression. A rather novel development is the use of lentivectors to transfer transgenes in oocytes and early embryos to generate transgenic animals, a technology also known as lentiviral transgenesis. Lentiviral transgenesis has been shown to be highly efficient in many different species, including mouse, rat, pig, bovine, and even birds. Thus, lentiviral transgenesis has the potential to become a versatile and widespread transgenic technology. The aim of this chapter is to cover important practical aspects of lentiviral transgenesis, including vector preparation, gene delivery into the early embryos and lentiviral RNA interference.

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References

  1. Jaenisch R, Mintz B. Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc Natl Acad Sci USA 1974; 71:1250–4.

    Article  PubMed  CAS  Google Scholar 

  2. Jaenisch R, Fan H, Croker B. Infection of preimplantation mouse embryos and of newborn mice with leukemia virus: tissue distribution of viral DNA and RNA and leukemogenesis in the adult animal. Proc Natl Acad Sci USA 1975; 72:4008–12.

    Article  PubMed  CAS  Google Scholar 

  3. Goff SP. Retroviridae: The retroviruses and their replication. In: Howley PM, Knipe DM, Griffin D, et al., eds. Fields Virology. Vol. 2 (4th ed.). Philadelphia: Lippincott-Raven Publishers. 2001; 1871–1939. 2001.

    Google Scholar 

  4. Jaenisch R. Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc Natl Acad Sci USA 1976; 73:1260–4.

    Article  PubMed  CAS  Google Scholar 

  5. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 1980; 77:7380–4.

    Article  PubMed  CAS  Google Scholar 

  6. Chan AW, Homan EJ, Ballou LU, Burns JC, Bremel RD. Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proc Natl Acad Sci USA 1998; 95:14028–33.

    Article  PubMed  CAS  Google Scholar 

  7. Pfeifer A. Lentiviral transgenesis. Transgenic Res 2004; 13:513–22.

    Article  PubMed  CAS  Google Scholar 

  8. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 2002; 295:868–72.

    Article  PubMed  CAS  Google Scholar 

  9. Pfeifer A, Ikawa M, Dayn Y, Verma IM. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci USA 2002; 99:2140–5.

    Article  PubMed  CAS  Google Scholar 

  10. Hofmann A, Kessler B, Ewerling S, et al. Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep 2003; 4:1054–60.

    Article  PubMed  CAS  Google Scholar 

  11. Whitelaw CB, Radcliffe PA, Ritchie WA, et al. Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector. FEBS Lett 2004; 571:233–6.

    Article  PubMed  CAS  Google Scholar 

  12. Hofmann A, Zakhartchenko V, Weppert M, et al. Generation of transgenic cattle by lentiviral gene transfer into oocytes. Biol Reprod 2004; 71:405–9.

    Article  PubMed  CAS  Google Scholar 

  13. McGrew MJ, Sherman A, Ellard FM, et al. Efficient production of germline transgenic chickens using lentiviral vectors. EMBO Rep 2004; 5:728–33.

    Article  PubMed  CAS  Google Scholar 

  14. Scott BB, Lois C. Generation of tissue-specific transgenic birds with lentiviral vectors. Proc Natl Acad Sci USA 2005; 102:16443–7.

    Article  PubMed  CAS  Google Scholar 

  15. Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998; 72:8463–71.

    PubMed  CAS  Google Scholar 

  16. Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. Development of a self-inactivating lentivirus vector. J Virol 1998; 72:8150–7.

    PubMed  CAS  Google Scholar 

  17. Nagy A, Gertsenstein M, Vintersten K, Behringer R. Manipulating the Mouse Embryo A Laboratory Manual. 3rd edition, Cold Spring Harbor Press. 2003.

    Google Scholar 

  18. Hannon GJ, Rossi JJ. Unlocking the potential of the human genome with RNA interference. Nature 2004; 431:371–8.

    Article  PubMed  CAS  Google Scholar 

  19. Novina CD, Sharp PA. The RNAi revolution. Nature 2004; 430:161–4.

    Article  PubMed  CAS  Google Scholar 

  20. Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002; 20:500–5.

    PubMed  CAS  Google Scholar 

  21. Tiscornia G, Singer O, Ikawa M, Verma IM. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci USA 2003; 100:1844–8.

    Article  PubMed  CAS  Google Scholar 

  22. Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 2003; 33:401–6.

    Article  PubMed  CAS  Google Scholar 

  23. Singer O, Marr RA, Rockenstein E, et al. Targeting BACE1 with siRNAs ameliorates Alzheimers disease neuropathology in a transgenic model. Nat Neurosci 2005; 8:1343–9.

    Article  PubMed  CAS  Google Scholar 

  24. Pfeifer A, Eigenbrod S, Al-Khadra S, et al. Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice. J Clin Invest 2006; 116:3204–10.

    Article  PubMed  CAS  Google Scholar 

  25. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol 2004; 22:326–30.

    Article  PubMed  CAS  Google Scholar 

  26. Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003; 115:199–208.

    Article  PubMed  CAS  Google Scholar 

  27. Heale BS, Soifer HS, Bowers C, Rossi JJ. siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res 2005; 33:e30.

    Article  PubMed  Google Scholar 

  28. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296:550–3.

    Article  PubMed  CAS  Google Scholar 

  29. Boden D, Pusch O, Lee F, Tucker L, Shank PR, Ramratnam B. Promoter choice affects the potency of HIV-1 specific RNA interference. Nucleic Acids Res 2003; 31:5033–8.

    Article  PubMed  CAS  Google Scholar 

  30. Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 1996; 93:11382–8.

    Article  PubMed  CAS  Google Scholar 

  31. Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 2000; 25:217–22.

    Article  PubMed  CAS  Google Scholar 

  32. Hornung V, Guenthner-Biller M, Bourquin C, et al. Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med 2005; 11:263–70.

    Article  PubMed  CAS  Google Scholar 

  33. Robbins MA, Li M, Leung I, et al. Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro. Nat Biotechnol 2006; 24:566–71.

    Article  PubMed  CAS  Google Scholar 

  34. Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 2003; 34:263–4.

    Article  PubMed  CAS  Google Scholar 

  35. Furumoto Y, Brooks S, Olivera A, et al. Cutting Edge: Lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival. J Immunol 2006; 176:5167–71.

    PubMed  CAS  Google Scholar 

  36. Pfeifer A, Verma IM. Gene therapy: promises and problems. Annu Rev Genomics Hum Genet 2001; 2:177–211.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors would like to thank Dr. Michael Bösl (Head of Group Transgenic Service, Max Planck Institute of Neurobiology, Martinsried) for providing embryos, culture media and valuable tips for embryo manipulation. This work was supported by the DFG and by Bonfor.

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Pfeifer, A., Hofmann, A. (2009). Lentiviral Transgenesis. In: Wurst, W., Kühn, R. (eds) Gene Knockout Protocols. Methods in Molecular Biology, vol 530. Humana Press. https://doi.org/10.1007/978-1-59745-471-1_21

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  • DOI: https://doi.org/10.1007/978-1-59745-471-1_21

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-26-8

  • Online ISBN: 978-1-59745-471-1

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