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.

  • Article
  • Published:

Streptogramin-based gene regulation systems for mammalian cells

Nature Biotechnology volume 18, pages 1203–1208 (2000)Cite this article

Abstract

Here we describe repressible (PipOFF) as well as inducible (PipON) systems for regulated gene expression in mammalian cells, based on the repressor Pip (pristinamycin-induced protein), which is encoded by the streptogramin resistance operon of Streptomyces coelicolor. Expression of genes placed under control of these systems was responsive to clinically approved antibiotics belonging to the streptogramin group (pristinamycin, virginiamycin, and Synercid). The versatility of these systems was demonstrated by streptogramin-regulated expression of mouse erythropoietin (EPO), human placental secreted alkaline phosphatase (SEAP), or green fluorescent protein (GFP) in diverse cell lines (BHK, CHO, HeLa, and mouse myoblasts). Analysis of isogenic constructs in CHO cells demonstrated the PipOFF system gave lower background and higher induction ratios than the widely used tetracycline-repressible (TetOFF) expression systems. The streptogramin-based expression technology was functionally compatible with the TetOFF system, thus enabling the selective use of different antibiotics to independently control two different gene activities in the same cell.

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: Gel retardation assay showing Pip–PPTR interaction.
Figure 2: Regulation of gene expression by streptogramin antibiotics.
Figure 3: Dose–response curve for PI-dependent gene expression.
Figure 4: Autoregulation and dual-regulation concepts.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Fussenegger, M., Schlatter, S., Dätwyler, D., Mazur, X. & Bailey, J.E. Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat. Biotechnol. 16, 468– 472 (1998).

    Article  CAS  Google Scholar 

  3. Umaña, P., Jean-Mairet, J. & Bailey, J.E. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 17, 176–180 (1999).

    Article  Google Scholar 

  4. Bohl, D., Naffakh, N. & Heard, J.M. Modulation of erythropoietin delivery from engineered muscles in mice. Hum. Gene Ther. 8, 195– 201 (1997).

    Article  CAS  Google Scholar 

  5. Serguera, C., Bohl, D., Rolland, E., Prevost, P. & Heard, J.M. Control of erythropoietin secretion by doxycycline or mifepristone in mice bearing polymer-encapsulated engineered cells. Hum. Gene Ther. 19, 375– 383 (1999).

    Article  Google Scholar 

  6. Rossi, F.M.V. et al. Tetracycline-regulated factors with distinct dimerization domains allow reversible growth inhibition by p16. Nat. Genet. 20, 389–393 (1998).

    Article  CAS  Google Scholar 

  7. Baim, S.B., Labow, M.A., Levine, A.J. & Shenk, T.A. Chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropylbeta-D-thiogalactopyranoside. Proc. Natl. Acad. Sci. USA 88, 5072–5076 (1991).

    Article  CAS  Google Scholar 

  8. Braselmann, S., Graninger, P. & Busslinger, M. A selective transcriptional induction system for mammalian cells based on Gal4-estrogen receptor fusion proteins. Proc. Natl. Acad. Sci. USA 90, 1657– 1661 (1993).

    Article  CAS  Google Scholar 

  9. No, D., Yao, T.-P. & Evans R.M. Ecdysone-inducible gene expression system in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA 93, 3346–3351 (1996).

    Article  CAS  Google Scholar 

  10. Rivera, V.M. et al. A humanized system for pharmacologic control of gene expression. Nat. Med. 2, 1028–1032 ( 1996).

    Article  CAS  Google Scholar 

  11. Suhr, S.T., Gil, E.B., Senut, M.-C. & Gage, F.H. High level transactivation by a modified Bombyx ecdysone receptor in mammalian cells without exogenous retinoid X receptor. Proc. Natl. Acad. Sci. USA 95, 7999–8004 (1998).

    Article  CAS  Google Scholar 

  12. Wang Y., O'Malley Jr., B.W., Tsai, S. & O'Malley, B. A regulatory system for use in gene transfer. Proc. Natl. Acad. Sci. USA 91, 8180–8184 ( 1994).

    Article  CAS  Google Scholar 

  13. Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769 ( 1995).

    Article  CAS  Google Scholar 

  14. Barrière, J.C., Bouanchaud, D.H., Desnottes, J.F. & Paris, J.M. Streptogramin analogues. Expert Opin. Invest. Drugs 3, 115–131 ( 1994).

    Article  Google Scholar 

  15. Baquero, F. Gram-positive resistance: challenge for the development of new antibiotics . J. Antimicrob. Chemother. 39, 1– 6 (1997).

    Article  CAS  Google Scholar 

  16. Blanc, V., Salah-Bey, K., Folcher, M. & Thompson, C.J. Molecular characterization and transcriptional analysis of a multidrug resistance gene cloned from the pristinamycin-producing organism, Streptomyces pristinaespiralis . Mol. Microbiol. 17, 989– 999 (1995).

    Article  CAS  Google Scholar 

  17. Salah-Bey, K., Blanc, V. & Thompson, C.J. Stress-activated expression of a Streptomyces pristinaespiralis multidrug resistance gene (ptr) in various Streptomyces spp. and Escherichia coli. Mol. Microbiol. 17, 1001–1012 (1995).

    Article  CAS  Google Scholar 

  18. Salah-Bey, K. & Thompson, C.J. Unusual regulatory mechanism for a Streptomyces multidrug resistance gene, ptr, involving three homologous protein-binding sites overlapping the promoter region. Mol. Microbiol. 17, 1109– 1119 (1995).

    Article  CAS  Google Scholar 

  19. Triezenberg, S.J., Kingsbury, R.C. & McKnight, S.L. Functional dissection of VP16, the trasactivator of herpes simplex virus immediate early gene expression . Genes Dev. 2, 718–729 (1988).

    Article  CAS  Google Scholar 

  20. Corces, V. & Pellicer, A. Identification of sequences involved in the transcriptional control of a Drosophila heat-shock gene. J. Biol. Chem. 259, 14812– 14817 (1984).

    CAS  PubMed  Google Scholar 

  21. Hillen, W. & Behrens, C. Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu. Rev. Microbiol. 48, 345–369 ( 1994).

    Article  CAS  Google Scholar 

  22. Bébéar, C. & Bouanchaud, D.H. A review of the in-vitro activity of quinupristin/dalfopristin against intracellular pathogens and mycoplasmas. J. Antimicrob. Chemother. 39, 59–62 (1997).

    Article  Google Scholar 

  23. Fussenegger, M., Moser, S., Mazur, X. & Bailey, J.E. Autoregulated multicistronic expression vectors provide one-step cloning of regulated product gene expression in mammalian cells. Biotechnol. Prog. 13, 733–740 ( 1997).

    Article  CAS  Google Scholar 

  24. Bessis, N. et al. Encapsulation in hollow fibres of xenogeneic cells engineered to secrete IL-4 or IL-13 ameliorates murine collagen-induced arthritis (CIA). Clin. Exp. Immunol. 117, 376–382 (1999).

    Article  CAS  Google Scholar 

  25. Dalle, B. et al. Improvement of mouse beta-thalassemia upon erythropoietin delivery by encapsulated myoblasts. Gene Ther. 6, 157– 161 (1999).

    Article  CAS  Google Scholar 

  26. Regulier, E., Schneider, B.L., Deglon, N., Beuzard, Y. & Aebischer, P. Continuous delivery of human and mouse erythropoietin in mice by genetically engineered polymer encapsulated myoblasts. Gene Ther. 5, 1014 –1022 (1998).

    Article  CAS  Google Scholar 

  27. Moosmann, P., Georgiev, O., Thiesen, H.J., Hagmann, M. & Schaffner, W. Silencing of RNA polymerases II and III-dependent transcription by the KRAB protein domain of Kox1, a kruppel-type zinc finger factor. Biol. Chem. 387, 669 –677 (1997).

    Google Scholar 

  28. Bergeron, M. & Montay, G. The pharmacokinetics of quinupristin/dalfopristin in laboratory animals and humans. J. Antimicrob. Chemother. 39, 129– 138 (1997).

    Article  CAS  Google Scholar 

  29. Baron, U. et al. Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes. Proc. Natl. Acad. Sci. USA 96, 1013–1018 (1999).

    Article  CAS  Google Scholar 

  30. Fussenegger, M., Mazur, X. & Bailey, J.E. pTRIDENT, a novel vector family for tricistronic gene expression in mammalian cells. Biotechnol. Bioeng. 57, 1–10 (1998).

    Article  CAS  Google Scholar 

  31. Fussenegger, M., Mazur, X. & Bailey, J.E. A novel cytostatic process enhances the productivity of Chinese hamster ovary cells. Biotechnol. Bioeng. 55, 927–939 ( 1997).

    Article  CAS  Google Scholar 

  32. Berger, J., Hauber, J., Hauber, R., Geiger, R. & Cullen, B.R. Secreted alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66, 1–10 ( 1988).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank U. Billeter for providing pristinamycin and its derivatives, E. Niederer for FACS sorting, S. Moser for construction of pDuoRex1 precursors, P. Aebischer for EPO expression construct pM48cDNAmEPO, and P. Viollier, M. Folcher, and U. Billeter for their generous advice during the project. This work was supported by Cistronics Cell Technology GmbH (Einsteinstrasse, P.O. Box 145, CH-8093 Zurich, Switzerland).

Author information

Author notes

  1. Charles J. Thompson and James E. Bailey: Both authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich, CH-8093, Zurich , Switzerland

    Martin Fussenegger, Cornelia Fux, Markus Rimann, Beryl von Stockar & James E. Bailey

  2. Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel , CH-4056, Switzerland

    Rowan P. Morris & Charles J. Thompson

Authors
  1. Martin Fussenegger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rowan P. Morris
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cornelia Fux
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Markus Rimann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Beryl von Stockar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Charles J. Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. James E. Bailey
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martin Fussenegger.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fussenegger, M., Morris, R., Fux, C. et al. Streptogramin-based gene regulation systems for mammalian cells. Nat Biotechnol 18, 1203–1208 (2000). https://doi.org/10.1038/81208

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/81208

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing