Skip to main content

Advertisement

SpringerLink
Log in

Variegation and silencing in a lentiviral-based murine transgenic model

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

Lentiviral based constructs represent a recent development in the generation of transgenic animals. The ease of use, and the fact that the same backbone vectors can be used to down-modulate endogenous gene expression and to produce transgenic animals overexpressing a gene of interest, have fuelled growing interest in this technology. In this study, we have used a lentiviral delivery system to generate transgenic mice expressing altered levels (up or downregulated) of a gene of interest. Although this lentiviral-based approach led to high levels of transgenesis and germ line transmission, a wide variation in transgene expression was observed in most first and second generation mouse lines. In particular, despite the segregation of integrants into single-copy expressing mouse lines, transgene expression appeared to be the target of epigenetic regulatory mechanism, often causing the coexistence of high and low transgene expressing cells within a given tissue such as blood peripheral lymphocytes. The establishment and analysis of large number of mouse lines may therefore be required to select a stable transgenic line with pancellular expression of a gene of interest using this lentiviral-based approach.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Bednarik DP, Cook JA, Pitha PM (1990) Inactivation of the HIV LTR by DNA CpG methylation: evidence for a role in latency. EMBO J 9:1157–1164

    CAS  PubMed  Google Scholar 

  • Breckpot K, Dullaers M, Bonehill A, van Meirvenne S, Heirman C, de Greef C, van der Bruggen P, Thielemans K (2003) Lentivirally transduced dendritic cells as a tool for cancer immunotherapy. J Gene Med 5:654–667

    Article  CAS  PubMed  Google Scholar 

  • Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553

    Article  CAS  PubMed  Google Scholar 

  • Dann CT (2007) New technology for an old favorite: lentiviral transgenesis and RNAi in rats. Transgenic Res 16:571–580

    Article  CAS  PubMed  Google Scholar 

  • Dann CT, Garbers DL (2008) Production of knockdown rats by lentiviral transduction of embryos with short hairpin RNA transgenes. Methods Mol Biol 450:193–209

    Article  CAS  PubMed  Google Scholar 

  • Dobie K, Mehtali M, McClenaghan M, Lathe R (1997) Variegated gene expression in mice. Trends Genet 13:127–130

    Article  CAS  PubMed  Google Scholar 

  • Ellis J (2005) Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum Gene Ther 16:1241–1246

    Article  CAS  PubMed  Google Scholar 

  • Fregien N, Davidson N (1986) Activating elements in the promoter region of the chicken beta-actin gene. Gene 48:1–11

    Article  CAS  PubMed  Google Scholar 

  • Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 77:7380–7384

    Article  CAS  PubMed  Google Scholar 

  • Hamra FK, Gatlin J, Chapman KM, Grellhesl DM, Garcia JV, Hammer RE, Garbers DL (2002) Production of transgenic rats by lentiviral transduction of male germ-line stem cells. Proc Natl Acad Sci USA 99:14931–14936

    Article  CAS  PubMed  Google Scholar 

  • Hannon GJ (2002) RNA interference. Nature 418:244–251

    Article  CAS  PubMed  Google Scholar 

  • Hofmann A, Kessler B, Ewerling S, Weppert M, Vogg B, Ludwig H, Stojkovic M, Boelhauve M, Brem G, Wolf E, Pfeifer A (2003) Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep 4:1054–1060

    Article  CAS  PubMed  Google Scholar 

  • Hofmann A, Kessler B, Ewerling S, Kabermann A, Brem G, Wolf E, Pfeifer A (2006) Epigenetic regulation of lentiviral transgene vectors in a large animal model. Mol Ther 13:59–66

    Article  CAS  PubMed  Google Scholar 

  • Ikawa M, Tanaka N, Kao WW, Verma IM (2003) Generation of transgenic mice using lentiviral vectors: a novel preclinical assessment of lentiviral vectors for gene therapy. Mol Ther 8:666–673

    Article  CAS  PubMed  Google Scholar 

  • Karkkainen AM, Kotimaa A, Huusko J, Kholova I, Heinonen SE, Stefanska A, Dijkstra MH, Purhonen H, Hamalainen E, Makinen PI, Turunen MP, Yla-Herttuala S (2009) VEGF-D transgenic mice show enhanced blood capillary density, improved post-ischemic muscle regeneration and increased susceptibility to tumor formation. Blood 113:4468–4475

    Article  CAS  PubMed  Google Scholar 

  • Kirilov M, Chai M, van der Hoeven F, Kloz U, Schmid W, Schutz G (2007) Germ line transmission and expression of an RNAi cassette in mice generated by a lentiviral vector system. Transgenic Res 16:783–793

    Article  CAS  PubMed  Google Scholar 

  • Kissler S, Stern P, Takahashi K, Hunter K, Peterson LB, Wicker LS (2006) In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes. Nat Genet 38:479–483

    Article  CAS  PubMed  Google Scholar 

  • Kvell K, Czompoly T, Hiripi L, Balogh P, Kobor J, Bodrogi L, Pongracz JE, Ritchie WA, Bosze Z (2009) Characterisation of eGFP-transgenic BALB/c mouse strain established by lentiviral transgenesis. Transgenic Res. doi:10.1007/s11248-009-9288-6

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

    Article  CAS  PubMed  Google Scholar 

  • McGrew MJ, Sherman A, Ellard FM, Lillico SG, Gilhooley HJ, Kingsman AJ, Mitrophanous KA, Sang H (2004) Efficient production of germline transgenic chickens using lentiviral vectors. EMBO Rep 5:728–733

    Article  CAS  PubMed  Google Scholar 

  • Michalkiewicz M, Michalkiewicz T, Geurts AM, Roman RJ, Slocum GR, Singer O, Weihrauch D, Greene AS, Kaldunski M, Verma IM, Jacob HJ, Cowley AW Jr (2007) Efficient transgenic rat production by a lentiviral vector. Am J Physiol Heart Circ Physiol 293:H881–H894

    Article  CAS  PubMed  Google Scholar 

  • Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM (1998) Development of a self-inactivating lentivirus vector. J Virol 72:8150–8157

    CAS  PubMed  Google Scholar 

  • Naldini L, Blomer U, Gage FH, Trono D, Verma IM (1996a) 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 93:11382–11388

    Article  CAS  PubMed  Google Scholar 

  • Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D (1996b) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263–267

    Article  CAS  PubMed  Google Scholar 

  • Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199

    Article  CAS  PubMed  Google Scholar 

  • Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407:313–319

    Article  CAS  PubMed  Google Scholar 

  • Oldenhove G, de Heusch M, Urbain-Vansanten G, Urbain J, Maliszewski C, Leo O, Moser M (2003) CD4 + CD25 + regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo. J Exp Med 198:259–266

    Article  CAS  PubMed  Google Scholar 

  • Onnockx S, De Schutter J, Blockmans M, Xie J, Jacobs C, Vanderwinden JM, Erneux C, Pirson I (2008) The association between the SH2-containing inositol polyphosphate 5-Phosphatase 2 (SHIP2) and the adaptor protein APS has an impact on biochemical properties of both partners. J Cell Physiol 214:260–272

    Article  CAS  PubMed  Google Scholar 

  • Pannell D, Ellis J (2001) Silencing of gene expression: implications for design of retrovirus vectors. Rev Med Virol 11:205–217

    Article  CAS  PubMed  Google Scholar 

  • Park F (2007) Lentiviral vectors: are they the future of animal transgenesis? Physiol Genomics 31:159–173

    Article  CAS  PubMed  Google Scholar 

  • Pfeifer A (2006) Lentiviral transgenesis–a versatile tool for basic research and gene therapy. Curr Gene Ther 6:535–542

    Article  CAS  PubMed  Google Scholar 

  • Pfeifer A, Hofmann A (2009) Lentiviral transgenesis. Methods Mol Biol 530:391–405

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, Rooney DL, Zhang M, Ihrig MM, McManus MT, Gertler FB, Scott ML, Van Parijs L (2003) 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

    Article  CAS  PubMed  Google Scholar 

  • Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F (2002) HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110:521–529

    Article  CAS  PubMed  Google Scholar 

  • Singer O, Verma IM (2008) Applications of lentiviral vectors for shRNA delivery and transgenesis. Curr Gene Ther 8:483–488

    Article  CAS  PubMed  Google Scholar 

  • Singer O, Tiscornia G, Ikawa M, Verma IM (2006) Rapid generation of knockdown transgenic mice by silencing lentiviral vectors. Nat Protoc 1:286–292

    Article  CAS  PubMed  Google Scholar 

  • Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ (2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA 102:13212–13217

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Van Laethem F, Baus E, Smyth LA, Andris F, Bex F, Urbain J, Kioussis D, Leo O (2001) Glucocorticoids attenuate T cell receptor signaling. J Exp Med 193:803–814

    Article  PubMed  Google Scholar 

  • Ventura A, Meissner A, Dillon CP, McManus M, Sharp PA, Van Parijs L, Jaenisch R, Jacks T (2004) Cre-lox-regulated conditional RNA interference from transgenes. Proc Natl Acad Sci USA 101:10380–10385

    Article  CAS  PubMed  Google Scholar 

  • Weinberger LS, Burnett JC, Toettcher JE, Arkin AP, Schaffer DV (2005) Stochastic gene expression in a lentiviral positive-feedback loop: HIV-1 Tat fluctuations drive phenotypic diversity. Cell 122:169–182

    Article  CAS  PubMed  Google Scholar 

  • Xia XG, Zhou H, Samper E, Melov S, Xu Z (2006) Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice. PLoS Genet 2:e10

    Article  PubMed  Google Scholar 

  • Yang SH, Cheng PH, Sullivan RT, Thomas JW, Chan AW (2008) Lentiviral integration preferences in transgenic mice. Genesis 46:711–718

    Article  CAS  PubMed  Google Scholar 

  • Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72:9873–9880

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank, M. Blockmans for providing lentiviral construct, T. Van Reeth for kind gift of the PGK-Cre expressing mouse strain. We also wish to thank Dr. D. Trono (University of Geneva, Switzerland) for providing the pWPXLd, psPAX2 and pMD2.G plasmids and Dr. SJ. Elledge (Harvard Medical School Boston, U.S.A.) for generously providing the pPRIME-CMV-GFP-FF3 vector. This work was supported by The Belgian Program in Interuniversity Poles of Attraction Initiated by the BelgianSate, Prime Minister’s office, Science Policy Programming, by a Research Concerted Action of the Communauté française de Belgique and by the “Région Wallonne” (Programme CIBLE), Belgium. DB, LF and EP were supported by a grant from “Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture, FRIA”, while SS and MM are supported by the “Fonds National de la Recherche Scientifique de Belgique, FRS-FNRS”. The scientific responsibility is assumed by the authors.

Author information

Authors and Affiliations

  1. Laboratoire de Physiologie Animale, Faculté des Sciences, Institut de Biologie et de Médecine Moléculaire, Université Libre de Bruxelles, Gosselies, Belgium

    Delphine Baup, Laurent Fraga, Annette Van Acker, Anne-Sophie Vanherck, Muriel Moser & Oberdan Leo

  2. Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Faculté de Médecine, Université Libre de Bruxelles, Gosselies, Belgium

    Eileen Pernot & Stéphane Schurmans

  3. Laboratory of Molecular and Cellular Therapy, Department of Physiology and Immunology, Medical School of the Vrije Universiteit Brussel, Brussel, Belgium

    Karine Breckpot & Kris Thielemans

Authors
  1. Delphine Baup
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laurent Fraga
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eileen Pernot
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Annette Van Acker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anne-Sophie Vanherck
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Karine Breckpot
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kris Thielemans
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stéphane Schurmans
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Muriel Moser
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Oberdan Leo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Oberdan Leo.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(PPT 1140 kb)

(PPT 296 kb)

(PPT 583 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Baup, D., Fraga, L., Pernot, E. et al. Variegation and silencing in a lentiviral-based murine transgenic model. Transgenic Res 19, 399–414 (2010). https://doi.org/10.1007/s11248-009-9318-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-009-9318-4

Keywords

Search

Navigation