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.

  • Original Article
  • Published:

Optimization of the Tet-On system for regulated gene expression through viral evolution

Gene Therapy volume 13, pages 1382–1390 (2006)Cite this article

Abstract

The ability to control (trans)gene expression is important both for basic biological research and applications such as gene therapy. In vivo use of the inducible tetracycline (Tc)-regulated gene expression system (Tet-On system) is limited by its low sensitivity for the effector doxycycline (dox). We used viral evolution to optimize this Escherichia coli-derived regulatory system for its function in mammalian cells. The components of the Tet-On system (the transcriptional activator rtTA and its tetO DNA binding site) were incorporated into the human immunodeficiency virus (HIV)-1 genome to control viral replication. Prolonged culturing of this HIV-rtTA virus resulted in virus variants that acquired mutations in the rtTA gene. Some of these mutations enhance the transcriptional activity and dox-sensitivity of the rtTA protein. This improvement was observed with different tetO-containing promoters and was independent of the episomal or chromosomal status of the target gene. Combination of these beneficial mutations resulted in greatly improved rtTA variants that are seven-fold more active and 100-fold more dox-sensitive than the original Tet-On system. Furthermore, some of the new Tet-On systems are responsive to Tc and minocycline. Importantly, these rtTA variants show no activity in the absence of dox. The optimized rtTA variants are particularly useful for in vivo applications that require a more sensitive or more active Tet-On system.

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. Fussenegger M . The impact of mammalian gene regulation concepts on functional genomic research, metabolic engineering, and advanced gene therapies. Biotechnol Prog 2001; 17: 1–51.

    Article  CAS  Google Scholar 

  2. Baron U, Bujard H . Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol 2000; 327: 401–421.

    Article  CAS  Google Scholar 

  3. Gossen M, Bujard H . Tetracyclines in the control of gene expression in eukaryotes. In: Nelson M, Hillen W, Greenwald RA (eds). Tetracyclines in Biology, Chemistry and Medicine. Birkhäuser Verlag: Basel, 2001, pp 139–157.

    Chapter  Google Scholar 

  4. Berens C, Hillen W . Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes. Eur J Biochem 2003; 270: 3109–3121.

    Article  CAS  Google Scholar 

  5. 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  Google Scholar 

  6. Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H . Transcriptional activation by tetracyclines in mammalian cells. Science 1995; 268: 1766–1769.

    Article  CAS  Google Scholar 

  7. Mohammadi S, Alvarez-Vallina L, Ashworth LJ, Hawkins RE . Delay in resumption of the activity of tetracycline-regulatable promoter following removal of tetracycline analogues. Gene Therapy 1997; 4: 993–997.

    Article  CAS  Google Scholar 

  8. Mizuguchi H, Hayakawa T . The tet-off system is more effective than the tet-on system for regulating transgene expression in a single adenovirus vector. J Gene Med 2002; 4: 240–247.

    Article  Google Scholar 

  9. Vogel R, Amar L, Thi AD, Saillour P, Mallet J . A single lentivirus vector mediates doxycycline-regulated expression of transgenes in the brain. Hum Gene Ther 2004; 15: 157–165.

    Article  CAS  Google Scholar 

  10. Baron U, Gossen M, Bujard H . Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. Nucleic Acids Res 1997; 25: 2723–2729.

    Article  CAS  Google Scholar 

  11. Baron U, Schnappinger D, Helbl V, Gossen M, Hillen W, Bujard H . Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes. Proc Natl Acad Sci USA 1999; 96: 1013–1018.

    Article  CAS  Google Scholar 

  12. Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W . Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 2000; 97: 7963–7968.

    Article  CAS  Google Scholar 

  13. Das AT, Zhou X, Vink M, Klaver B, Verhoef K, Marzio G et al. Viral evolution as a tool to improve the tetracycline-regulated gene expression system. J Biol Chem 2004; 279: 18776–18782.

    Article  CAS  Google Scholar 

  14. Verhoef K, Marzio G, Hillen W, Bujard H, Berkhout B . Strict control of human immunodeficiency virus type 1 replication by a genetic switch: Tet for Tat. J Virol 2001; 75: 979–987.

    Article  CAS  Google Scholar 

  15. Das AT, Verhoef K, Berkhout B . A conditionally replicating virus as a novel approach toward an HIV vaccine. Methods Enzymol 2004; 388: 359–379.

    Article  CAS  Google Scholar 

  16. Marzio G, Verhoef K, Vink M, Berkhout B . In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies. Proc Natl Acad Sci USA 2001; 98: 6342–6347.

    Article  CAS  Google Scholar 

  17. Marzio G, Vink M, Verhoef K, de Ronde A, Berkhout B . Efficient human immunodeficiency virus replication requires a fine-tuned level of transcription. J Virol 2002; 76: 3084–3088.

    Article  CAS  Google Scholar 

  18. Smith SM, Khoroshev M, Marx PA, Orenstein J, Jeang KT . Constitutively dead, conditionally live HIV-1 genomes. Ex vivo implications for a live virus vaccine. J Biol Chem 2001; 276: 32184–32190.

    Article  CAS  Google Scholar 

  19. Berkhout B, Verhoef K, Marzio G, Klaver B, Vink M, Zhou X et al. Conditional virus replication as an approach to a safe live attenuated human immunodeficiency virus vaccine. J Neurovirol 2002; 8 (Suppl 2): 134–137.

    Article  CAS  Google Scholar 

  20. Das AT, Zhou X, Vink M, Klaver B, Berkhout B . Conditional live virus as a novel approach towards a safe live attenuated HIV vaccine. Expert Rev Vaccines 2002; 1: 293–301.

    Article  CAS  Google Scholar 

  21. Hinrichs W, Kisker C, Duvel M, Muller A, Tovar K, Hillen W et al. Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance. Science 1994; 264: 418–420.

    Article  CAS  Google Scholar 

  22. Kisker C, Hinrichs W, Tovar K, Hillen W, Saenger W . The complex formed between Tet repressor and tetracycline-Mg2+ reveals mechanism of antibiotic resistance. J Mol Biol 1995; 247: 260–280.

    Article  CAS  Google Scholar 

  23. Orth P, Cordes F, Schnappinger D, Hillen W, Saenger W, Hinrichs W . Conformational changes of the Tet repressor induced by tetracycline trapping. J Mol Biol 1998; 279: 439–447.

    Article  CAS  Google Scholar 

  24. Orth P, Schnappinger D, Hillen W, Saenger W, Hinrichs W . Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nat Struct Biol 2000; 7: 215–219.

    Article  CAS  Google Scholar 

  25. Henssler EM, Scholz O, Lochner S, Gmeiner P, Hillen W . Structure-based design of Tet repressor to optimize a new inducer specificity. Biochemistry 2004; 43: 9512–9518.

    Article  CAS  Google Scholar 

  26. Scholz O, Kintrup M, Reich M, Hillen W . Mechanism of Tet repressor induction by tetracyclines: length compensates for sequence in the alpha8-alpha9 loop. J Mol Biol 2001; 310: 979–986.

    Article  CAS  Google Scholar 

  27. Muller G, Hecht B, Helbl V, Hinrichs W, Saenger W, Hillen W . Characterization of non-inducible Tet repressor mutants suggests conformational changes necessary for induction. Nat Struct Biol 1995; 2: 693–703.

    Article  CAS  Google Scholar 

  28. Schubert P, Schnappinger D, Pfleiderer K, Hillen W . Identification of a stability determinant on the edge of the Tet repressor four-helix bundle dimerization motif. Biochemistry 2001; 40: 3257–3263.

    Article  CAS  Google Scholar 

  29. Smith SD, Shatsky M, Cohen PS, Warnke R, Link MP, Glader BE . Monoclonal antibody and enzymatic profiles of human malignant T-lymphoid cells and derived cell lines. Cancer Res 1984; 44 (Part 1): 5657–5660.

    CAS  PubMed  Google Scholar 

  30. Auersperg N . Long-term cultivation of hypodiploid human tumor cells. J Natl Cancer Inst 1964; 32: 135–163.

    CAS  PubMed  Google Scholar 

  31. Das AT, Klaver B, Klasens BI, van Wamel JL, Berkhout B . A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication. J Virol 1997; 71: 2346–2356.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Mikaelian I, Sergeant A . A general and fast method to generate multiple site directed mutations. Nucleic Acids Res 1992; 20: 376.

    Article  CAS  Google Scholar 

  33. Das AT, Klaver B, Berkhout B . A hairpin structure in the R region of the human immunodeficiency virus type 1 RNA genome is instrumental in polyadenylation site selection. J Virol 1999; 73: 81–91.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Ruijter JM, Thygesen HH, Schoneveld OJ, Das AT, Berkhout B, Lamers WH . Factor correction as a tool to eliminate between-session variation in replicate experiments:application to molecular biology and retrovirology. Retrovirology 2006; 3: 2.

    Article  Google Scholar 

  35. Krueger C, Berens C, Schmidt A, Schnappinger D, Hillen W . Single-chain Tet transregulators. Nucleic Acids Res 2003; 31: 3050–3056.

    Article  CAS  Google Scholar 

  36. Kraulis PJ . MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 1991; 24: 946–950.

    Article  Google Scholar 

  37. Merritt EA, Bacon DJ . Raster3D: Photorealistic molecular graphics. Methods Enzymol 1997; 277: 505–524.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Rogier Sanders for help with the MOLSCRIPT and Raster3D programs, Jan M Ruyter (Department of Anatomy and Embryology, AMC) for help in data analysis, and Christel Krüger, Christian Berens and Wolfgang Hillen (University of Erlangen, Germany) for their generous gift of HeLa X1/6 cells, pUHC13-3, pCMV-rtTA, polyclonal anti-TetR antibodies and TetR protein. This research was sponsored by the Technology Foundation STW (Applied Science Division of NWO and the technology program of the Ministry of Economic Affairs, Utrecht, the Netherlands).

Author information

Authors and Affiliations

  1. Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

    X Zhou, M Vink, B Klaver, B Berkhout & A T Das

Authors
  1. X Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M Vink
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B Klaver
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. B Berkhout
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A T Das
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A T Das.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhou, X., Vink, M., Klaver, B. et al. Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Ther 13, 1382–1390 (2006). https://doi.org/10.1038/sj.gt.3302780

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302780

Keywords

This article is cited by

Search

Advanced search

Quick links