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Transgene Control Engineering in Mammalian Cells

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Therapeutic Proteins

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 308))

Abstract

Capitalizing on the generic design principle of the tetracycline-responsive expression systems (1,2), many various transgene control modalities responsive to streptogramins (3), macrolides (4), and butyrolactones (5) have been constructed. All of these gene-regulation systems could be arranged in small-molecule-repressible off-type and small-molecule-inducible on-type configurations. Off-type systems consist of a transactivator and transactivator-dependent promoter. The transactivator is a fusion protein between a bacterial small-molecule-responsive transcription regulator and mammalian transactivation domain, e.g., Herpes simplex-derived VP16 (6). The transactivator-dependent promoter consists of transactivator-specific (tandem) operator modules placed upstream of a minimal eukaryotic promoter, e.g., the minimal version of the human cytomegalovirus immediate early promoter (PhCVMmin). Transactivator binding to cognate operators in the absence of regulating small molecules initiates promoter-dependent transcription, whereas promoter-transactivator interaction is abolished in the presence of adjusting molecules, as is transgene expression (Fig. 1 A). On-type systems harbor the same prokaryotic response regulator that operates as a transrepressor on cognate chimeric promoters in its native form or optionally fused to a eukaryotic repression domain, such as the Kruppel-associated box (KRAB) domain of the human kox-1 gene (7). Transrepressor-dependent promoters consist of transrepressor-specific (tandem) operators cloned downstream of desired constitutive promoters. In the absence of regulating molecules, transrepressor-operator binding blocks/silences promoter-driven transgene expression, whereas small molecules interfering with transrepressor-operator interaction induce promoter-specific transcription (Fig. 1 B).

Molecular configuration of off-type (A) and on-type (B) gene regulatory systems. (A) off-type systems. A DNA-binding protein (DBP), typically a bacterial response regulator, fused to a mammalian transactivation domain (TA) binds to a specific operator module and induces polymerase (Poly)-mediated transcription of the gene of interest (goi) from a minimal promoter (Pmin). DBP-TA only binds to its operator in the absence of the regulating molecule (five-point star). Strategically important restriction sites are indicated. (B) on-type systems. The DBP, fused to a mammalian transcription repressor domain (TR), binds to the specific operator sequence located downstream of a constitutive promoter and thus represses its transcription. Upon addition of the regulating molecule, repression is abolished, and the gene of interest (goi) is expressed.

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References

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

    Article  PubMed  CAS  Google Scholar 

  2. Yao, F., Svensjo, T., Winkler, T., Lu, M., Eriksson, C., and Eriksson, E. (1998) Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Hum. Gene Ther. 9, 1939–1950.

    Article  PubMed  CAS  Google Scholar 

  3. Fussenegger, M., Morris, R. P., Fux, C., Rimann, M., von Stockar, B., Thompson, C. J., and Bailey, J. E. (2000) Streptogramin-based gene regulation systems for mammalian cells. Nat. Biotechnol. 18, 1203–1208.

    Article  PubMed  CAS  Google Scholar 

  4. Weber, W., Fux, C., Daoud-El Baba, M., Keller, B., Weber, C. C., Kramer, B. P., et al. (2002) Macrolide-based transgene control in mammalian cells and mice. Nat. Biotechnol. 20, 901–907.

    Article  PubMed  CAS  Google Scholar 

  5. Weber, W., Schoenmakers, R., Spielmann, M., Daoud-El Baba, M., Folcher, M., Keller, B., et al. (2003) Streptomyces-derived quorum-sensing systems engineered for adjustable transgene expression in mammalian cells and mice. Nucleic Acids Res. 31, e71.

    Article  PubMed  Google Scholar 

  6. Triezenberg, S. J., Kingsbury, R. C., and McKnight, S. L. (1988) Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 2, 718–729.

    Article  PubMed  CAS  Google Scholar 

  7. Moosmann, P., Georgiev, O., Thiesen, H. J., Hagmann, M., and Schaffner, W. (1997) Silencing of RNA polymerases II and III-dependent transcription by the KRAB protein domain of KOX1, a Kruppel-type zinc finger factor. Biol. Chem. 378, 669–677.

    Article  PubMed  CAS  Google Scholar 

  8. Fux, C. and Fussenegger, M. (2003) Bi-directional expression units enable streptogramin-adjustable gene expression in mammalian cells. Biotechnol. Bioeng. 83, 618–625.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  10. Moser, S., Rimann, M., Fux, C., Schlatter, S., Bailey, J. E., and Fussenegger, M. (2001) Dual-regulated expression technology: a new era in the adjustment of heterologous gene expression in mammalian cells. J. Gene Med. 3, 529–549.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  12. Rossi, F. M., Kringstein, A. M., Spicher, A., Guicherit, O. M., and Blau, H. M. (2000) Transcriptional control: rheostat converted to on/off switch. Mol. Cell. 6, 723–728.

    Article  PubMed  CAS  Google Scholar 

  13. Kramer, B. P., Fischer, C., and Fussenegger, M. (2004) BioLogic Gates enable logic transcription control in mammalian cells. Biotechnol. Bioeng. 87, 478–484.

    Article  PubMed  CAS  Google Scholar 

  14. Kramer, B. P., Viretta, A. U., Daoud-El-Baba, M., Aubel, D., Weber, W., and Fussenegger, M. (2004) An engineered epigenetic transgene switch in mammalian cells. Nat. Biotechnol. 22, 867–870.

    Article  PubMed  CAS  Google Scholar 

  15. Sambrook, J. and Russell, D. W. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.

    Google Scholar 

  16. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. Short Protocols in Molecular Biology. John Wiley & Sons, New York, NY, 1999.

    Google Scholar 

  17. Weber, W., Kramer, B. P., Fux, C., Keller, B., and Fussenegger, M. (2002) Novel promoter/transactivator configurations for macrolide-and streptogramin-responsive transgene expression in mammalian cells. J. Gene Med. 4, 676–686.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  19. Malphettes, L. and Fussenegger, M. (2004) Macrolide-and tetracycline-adjustable siRNA-mediated gene silencing in mammalian cells using polymerase II-dependent promoter derivatives. Biotechnol. Bioeng. 20, 417–425.

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  21. Grill, M. A., Bales, M. A., Fought, A. N., Rosburg, K. C., Munger, S. J., and Antin, P. B. (2003) Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice. Transgenic Res. 12, 33–43.

    Article  PubMed  CAS  Google Scholar 

  22. Imhof, M. O., Chatellard, P., and Mermod, N. (2002) Comparative study and identification of potent eukaryotic transcriptional repressors in gene switch systems. J. Biotechnol. 97, 275–285.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  24. Moser, S., Schlatter, S., Fux, C., Rimann, M., Bailey, J. E., and Fussenegger, M. (2000) An update of pTRIDENT multicistronic expression vectors: pTRIDENTs containing novel streptogramin-responsive promoters. Biotechnol. Prog. 16, 724–735.

    Article  PubMed  CAS  Google Scholar 

  25. Weber, W., Marty, R. R., Keller, B., Rimann, M., Kramer, B. P., and Fussenegger, M. (2002) Versatile macrolide-responsive mammalian expression vectors for multiregulated multigene metabolic engineering. Biotechnol. Bioeng. 80, 691–705.

    Article  PubMed  CAS  Google Scholar 

  26. Fux, C., Langer, D., Kelm, J. M., Weber, W., and Fussenegger, M. (2004) New-generation multicistronic expression platform: pTRIDENT vectors containing size-optimized IRES elements enable homing endonuclease-based cistron swapping into lentiviral expression vectors. Biotechnol. Bioeng. 86, 174–187.

    Article  PubMed  CAS  Google Scholar 

  27. Baron, U., Freundlieb, S., Gossen, M., and Bujard, H. (1995) Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res. 23, 3605–3606.

    Article  PubMed  CAS  Google Scholar 

  28. Fux, C., Weber, W., Daoud-El Baba, M., Heinzen, C., Aubel, D., and Fussenegger, M. (2003) Novel macrolide-adjustable bidirectional expression modules for coordinated expression of two different transgenes in mice. J. Gene Med. 5, 1067–1079.

    Article  PubMed  CAS  Google Scholar 

  29. Fux, C. and Fussenegger, M. (2003) Toward higher order control modalities in Mammalian cells-independent adjustment of two different gene activities. Biotechnol. Prog. 19, 109–120.

    Article  PubMed  CAS  Google Scholar 

  30. Mitta, B., Rimann, M., Ehrengruber, M. U., Ehrbar, M., Djonov, V., Kelm, J., and Fussenegger, M. (2002) Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction. Nucleic Acids Res. 30, e113.

    Article  PubMed  Google Scholar 

  31. Mitta, B., Weber, C. C., Rimann, M., and Fussenegger, M. (2004) Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression. Nucleic Acids Res. 32, e106.

    Article  PubMed  Google Scholar 

  32. Kramer, B. P., Weber, W., and Fussenegger, M. (2003) Artificial regulatory networks and cascades for discrete multilevel transgene control in mammalian cells. Biotechnol. Bioeng. 83, 810–820.

    Article  PubMed  CAS  Google Scholar 

  33. Gardner, T. S., Cantor, C. R., and Collins, J. J. (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Institute for Chemical and BioEngineering, Swiss Federal Institute of Technology, ETH Zurich, Zurich, Switzerland

    Beat P. Kramer & Martin Fussenegger

Authors
  1. Beat P. Kramer
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  2. Martin Fussenegger
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Editor information

Editors and Affiliations

  1. Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent, UK

    C. Mark Smales

  2. School of Engineering, University of Queensland, St. Lucia, Queensland, Australia

    David C. James

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© 2005 Humana Press Inc.

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Kramer, B.P., Fussenegger, M. (2005). Transgene Control Engineering in Mammalian Cells. In: Smales, C.M., James, D.C. (eds) Therapeutic Proteins. Methods in Molecular Biology™, vol 308. Humana Press. https://doi.org/10.1385/1-59259-922-2:123

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  • DOI: https://doi.org/10.1385/1-59259-922-2:123

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-390-9

  • Online ISBN: 978-1-59259-922-6

  • eBook Packages: Springer Protocols

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