Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS

Gene Therapy volume 9, pages 1291–1301 (2002)Cite this article

Abstract

Ex vivo gene transfer to the CNS has so far been hampered by instability of transgene expression. To avoid the phenomenon of transgene down-regulation, we have employed strong, constitutive promoters and compared this expression system with the inducible Tet expression system incorporated in a single plasmid vector or in lentiviral vectors. Plasmid-based transgene expression directed by the constitutive, human ubiquitin promoter, UbC, was stable in transfected HiB5 cells in vitro and comparable in strength to the CMV promoter. However, after transplantation of UbC and CMV HiB5 clones to the rat striatum, silencing of the transgene occurred in most cells soon after implantation of transfected cells. The Tet-on elements were incorporated in a single plasmid vector and inducible HiB5 clones were generated. Inducible clones displayed varying basal expression activity, which could not be ascribed to an effect of cis-elements in the vector, but rather was due, at least in part, to intrinsic activity of the minimal promoter. Basal expression activity could be blocked in a majority of cells by stable expressing the transrepressor tTS. Fully induced expression levels were comparable to CMV and UbC promoters. Similar to the constitutive promoters transgene expression was down-regulated soon after grafting of inducible HiB5 clones to the rat striatum. Lentiviral vectors can direct long-term stable in vivo transgene expression. To take advantage of this quality of the lentiviral vector, the Tet-on elements were incorporated in two lentiviral transfer vectors followed by transduction of Hib5 cells. Interestingly, all HiB5 clones established by lentiviral transduction showed very similar expression patterns and tight regulatability that apparently was independent of transgene copy number and integration site. Nevertheless, transgene expression in all lentiviral HiB5 clones was down-regulated shortly after transplantation to the rat striatum. These results confirm the general phenomenon of transgene down-regulation. Moreover, the results suggest that the considerable advantages offered by lentiviral vectors for direct gene delivery cannot necessarily be transferred directly to ex vivo gene delivery. This emphasizes the need for alternative vector strategies for ex vivo gene transfer.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Costantini LC, Bakowska JC, Breakefield XO, Isacson O . Gene therapy in the CNS Gene Therapy 2000 7: 93–109

    Article  CAS  PubMed  Google Scholar 

  2. Lundberg C, Horellou P, Mallet J, Bjorklund A . Generation of DOPA-producing astrocytes by retroviral transduction of the human tyrosine hydroxylase gene: in vitro characterization and in vivo effects in the rat Parkinson model Exp Neurol 1996 139: 39–53

    Article  CAS  PubMed  Google Scholar 

  3. Quinonero J et al. Gene transfer to the central nervous system by transplantation of cerebral endothelial cells Gene Therapy 1997 4: 111–119

    Article  CAS  PubMed  Google Scholar 

  4. Yoshimoto Y et al. Astrocytes retrovirally transduced with BDNF elicit behavioral improvement in a rat model of Parkinson's disease Brain Res 1995 691: 25–36

    Article  CAS  PubMed  Google Scholar 

  5. Schorpp M et al. The human ubiquitin C promoter directs high ubiquitous expression of transgenes in mice Nucleic Acids Res 1996 24: 1787–1788

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Johansen TE, Scholler MS, Tolstoy S, Schwartz TW . Biosynthesis of peptide precursors and protease inhibitors using new constitutive and inducible eukaryotic expression vectors FEBS Lett 1990 267: 289–294

    Article  CAS  PubMed  Google Scholar 

  7. Gossen M, Bujard H . Tight control of gene expression in mammalian cells by tetracycline-responsive promoters Proc Natl Acad Sci USA 1992 89: 5547–5551

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gossen M, Bujard H . Tight control of gene expression in mammalian cells by tetracycline-responsive promoters Hermann Bujard's Home Page:www.zmbh.uni-heidelberg.de/Bujard/Homepage.html.

  9. Kafri T, van Praag H, Gage FH, Verma IM . Lentiviral vectors: regulated gene expression Mol Ther 2000 1: 516–521

    Article  CAS  PubMed  Google Scholar 

  10. Blomer U et al. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector J Virol 1997 71: 6641–6649

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Harding TC et al. Switching transgene expression in the brain using an adenoviral tetracycline-regulatable system Nat Biotechnol 1998 16: 553–555

    Article  CAS  PubMed  Google Scholar 

  12. Bohl D, Naffakh N, Heard JM . Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts Nat Med 1997 3: 299–305

    Article  CAS  PubMed  Google Scholar 

  13. Renfranz PJ, Cunningham MG, McKay RD . Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain Cell 1991 66: 713–729

    Article  CAS  PubMed  Google Scholar 

  14. Englund U et al. The use of a recombinant lentiviral vector for ex vivo gene transfer into the rat CNS Neuroreport 2000 11: 3973–3977

    Article  CAS  PubMed  Google Scholar 

  15. Lundberg C et al. Survival, integration, and differentiation of neural stem cell lines after transplantation to the adult rat striatum Exp Neurol 1997 145: 342–360

    Article  CAS  PubMed  Google Scholar 

  16. Freundlieb S, Schirra-Muller C, Bujard H . A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells J Gene Med 1999 1: 4–12

    Article  CAS  PubMed  Google Scholar 

  17. Meyer M et al. Improved survival of embryonic porcine dopaminergic neurons in coculture with a conditionally immortalized GDNF-producing hippocampal cell line Exp Neurol 2000 164: 82–93

    Article  CAS  PubMed  Google Scholar 

  18. Clackson T . Regulated gene expression systems Gene Therapy 2000 7: 120–125

    Article  CAS  PubMed  Google Scholar 

  19. Gossen M et al. Transcriptional activation by tetracyclines in mammalian cells Science 1995 268: 1766–1769

    Article  CAS  PubMed  Google Scholar 

  20. Yin DX, Zhu L, Schimke RT . Tetracycline-controlled gene expression system achieves high-level and quantitative control of gene expression Anal Biochem 1996 235: 195–201

    Article  CAS  PubMed  Google Scholar 

  21. Garrick D, Fiering S, Martin DI, Whitelaw E . Repeat-induced gene silencing in mammals Nat Genet 1998 18: 56–59

    Article  CAS  PubMed  Google Scholar 

  22. Bujard H et al. Insertion of transcriptional elements outside the replication region can interfere with replication, maintenance, and stability of Co1E1-derived plasmids Basic Life Sci 1985 30: 45–52

    CAS  PubMed  Google Scholar 

  23. Hoffmann A, Villalba M, Journot L, Spengler D . A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammalian cell lines Nucleic Acids Res 1997 25: 1078–1079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Steinwaerder DS, Lieber A . Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo Gene Therapy 2000 7: 556–567

    Article  CAS  PubMed  Google Scholar 

  25. Martinez-Serrano A, Fischer W, Bjorklund A . Reversal of age-dependent cognitive impairments and cholinergic neuron atrophy by NGF-secreting neural progenitors grafted to the basal forebrain Neuron 1995 15: 473–484

    Article  CAS  PubMed  Google Scholar 

  26. Liu Y et al. Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function J Neurosci 1999 19: 4370–4387

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  28. Gao Z et al. High levels of transgene expression following transduction of long-term nod/scid-repopulating human cells with a modified lentiviral vector Stem Cells 2001 19: 247–259

    Article  CAS  PubMed  Google Scholar 

  29. Gallichan WS et al. Lentivirus-mediated transduction of islet grafts with interleukin 4 results in sustained gene expression and protection from insulitis Hum Gene Ther 1998 9: 2717–2726

    Article  CAS  PubMed  Google Scholar 

  30. Szyf M . The DNA methylation machinery as a target for anticancer therapy Pharmacol Ther 1996 70: 1–37

    Article  CAS  PubMed  Google Scholar 

  31. Siegfried Z et al. DNA methylation represses transcription in vivo Nat Genet 1999 22: 203–206

    Article  CAS  PubMed  Google Scholar 

  32. Chen WY et al. Reactivation of silenced, virally transduced genes by inhibitors of histone deacetylase Proc Natl Acad Sci USA 1997 94: 5798–5803

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tsai DJ, Ho JJ, Ozawa CR, Sapolsky RM . Long-term expression driven by herpes simplex virus type-1 amplicons may fail due to eventual degradation or extrusion of introduced transgenes Exp Neurol 2000 165: 58–65

    Article  CAS  PubMed  Google Scholar 

  34. Pikaart MJ, Recillas-Targa F, Felsenfeld G . Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators Genes Dev 1998 12: 2852–2862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Rivella S et al. The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites J Virol 2000 74: 4679–4687

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Chung JH, Whiteley M, Felsenfeld G . A 5’ element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila Cell 1993 74: 505–514

    Article  CAS  PubMed  Google Scholar 

  37. Jordan A, Defechereux P, Verdin E . The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation EMBO J 2001 20: 1726–1738

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zufferey R, Donello JE, Trono D, Hope TJ . Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors J Virol 1999 73: 2886–2892

    CAS  PubMed  PubMed Central  Google Scholar 

  39. von Schwedler U, Song J, Aiken C, Trono D . Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells J Virol 1993 67: 4945–4955

    CAS  PubMed  PubMed Central  Google Scholar 

  40. von Schwedler U, Song J, Aiken C, Trono D . Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells Brown T. Southern blotting. In: Ausubel et al (eds). Current Protocols in Molecular Biology. John Wiley & Sons: NY, 2001, pp 2.9.1–2.9.15.

  41. Rosenblad C et al. Protection and regeneration of nigral dopaminergic neurons by neurturin or GDNF in a partial lesion model of Parkinson's disease after administration into the striatum or the lateral ventricle Eur J Neurosci 1999 11: 1554–1566

    Article  CAS  PubMed  Google Scholar 

  42. Kirik D, Rosenblad C, Bjorklund A, Mandel RJ . Long-term rAAV-mediated gene transfer of GDNF in the rat Parkinson's model: intrastriatal but not intranigral transduction promotes functional regeneration in the lesioned nigrostriatal system J Neurosci 2000 20: 4686–4700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sauer H, Oertel WH . Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat Neuroscience 1994 59: 401–415

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We appreciate the excellent technical support from Birgitte Romme Larsen and Ulla Jarl. We thank D Trono for the generous gift of the lentiviral vector system.

Author information

Authors and Affiliations

  1. NsGene A/S, Ballerup, Denmark

    J Johansen, C Rosenblad, A Møller & T E Johansen

  2. Division of Neurobiology, Wallenberg Neuroscience Center, Lund University, Sölvegatan, Sweden

    K Andsberg, C Lundberg & A Björlund

Authors
  1. J Johansen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C Rosenblad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K Andsberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A Møller
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C Lundberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A Björlund
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. T E Johansen
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Johansen, J., Rosenblad, C., Andsberg, K. et al. Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS. Gene Ther 9, 1291–1301 (2002). https://doi.org/10.1038/sj.gt.3301778

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3301778

Keywords

This article is cited by

Search

Advanced search

Quick links