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Generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtTA2S-M2

Nature Protocols volume 1, pages 803–811 (2006)Cite this article

Abstract

Tet-on cell lines engineered to stably express doxycycline (Dox)-regulated reverse transcriptional transactivator (rtTA) have many applications in biomedical research and biotechnology. Unfortunately, construction and maintenance of such cells often proves to be costly, labor intensive and ineffective. Moreover, the Tet-on clones generated using standard methodology were often unstable and frequently displayed significantly changed physiological properties compared with their parental cells. Here we describe an optimized protocol for generation of Tet-on cells. The protocol is based on the use of a recently developed pN1pβactin-rtTA2S-M2-IRES-EGFP vector (where IRES is an internal ribosome entry site) and permits relatively inexpensive construction of many Tet-on clones with essentially 100% efficiency. The method is well suited for 'difficult' cell lines displaying genetic instability and high levels of epigenetic silencing. The constructed Tet-on cells remain stable with time in the absence of any selection agents, are easy to monitor and preserve the characteristics of parental cells. The protocol can be completed in 2 months.

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Figure 1: Summary of the principles and protocol for generation of Tet-on cells.
Figure 4
Figure 2: Selected properties of Tet-on cells established using this protocol.
Figure 3: Representative flow-cytometric profile of cells transfected with pN1pβactin-rtTA2S-M2-IRES-EGFP plasmid after G418 selection and growth for 7 d without G418 (using HCT116 cells).

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References

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

    Article  CAS  Google Scholar 

  2. Kaufmann, H. & Fussenegger, M. Metabolic engineering of mammalian cells for higher protein yieldin. Gene Transfer and Expression in Mammalian Cells (ed. Makrides, S.C.) 457–569 (Elsevier Science, Amsterdam, 2003).

    Chapter  Google Scholar 

  3. Toniatti, C., Bujard, H., Cortese, R. & Ciliberto, G. Gene therapy progress and prospects: transcription regulatory systems. Gene Ther. 11, 649–657 (2004).

    Article  CAS  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. 270, 3109–3121 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Corbel, S.Y. & Rossi, F.M. Latest developments and in vivo use of the Tet system: ex vivo and in vivo delivery of tetracycline-regulated genes. Curr. Opin. Biotechnol. 13, 448–452 (2002).

    Article  CAS  Google Scholar 

  7. Zhu, Z., Zheng, T., Lee, C.G., Homer, R.J. & Elias, J.A. Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling. Semin. Cell Dev. Biol. 13, 121–128 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Urlinger, S. et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. USA 97, 7963–7968 (2000).

    Article  CAS  Google Scholar 

  10. Hofmann, A., Nolan, G.P. & Blau, H.M. Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette. Proc. Natl. Acad. Sci. USA 93, 5185–5190 (1996).

    Article  CAS  Google Scholar 

  11. Chtarto, A. et al. Tetracycline-inducible transgene expression mediated by a single AAV vector. Gene Ther. 10, 84–94 (2003).

    Article  CAS  Google Scholar 

  12. Szulc, J., Wiznerowicz, M., Sauvain, M.O., Trono, D. & Aebischer, P. A versatile tool for conditional gene expression and knockdown. Nat. Methods 3, 109–116 (2006).

    Article  CAS  Google Scholar 

  13. Gopalkrishnan, R.V., Christiansen, K.A., Goldstein, N.I., DePinho, R.A. & Fisher, P.B. Use of the human EF-1α promoter for expression can significantly increase success in establishing stable cell lines with consistent expression: a study using the tetracycline-inducible system in human cancer cells. Nucleic Acids Res. 27, 4775–4782 (1999).

    Article  CAS  Google Scholar 

  14. Munoz, I., Gomez, A., Zanuy, S. & Carrillo, M. A one-step approach to obtain cell clones expressing tetracycline-responsive transactivators. Anal. Biochem. 331, 153–160 (2004).

    Article  CAS  Google Scholar 

  15. Whitelaw, E. et al. Epigenetic effects on transgene expression. Methods Mol. Biol. 158, 351–368 (2001).

    CAS  PubMed  Google Scholar 

  16. Welman, A. et al. Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression. J. Cell. Biochem. 94, 1148–1162 (2005).

    Article  CAS  Google Scholar 

  17. Qu, Z. et al. Homogeneity and long-term stability of tetracycline-regulated gene expression with low basal activity by using the rtTA2S-M2 transactivator and insulator-flanked reporter vectors. Gene 327, 61–73 (2004).

    Article  CAS  Google Scholar 

  18. Mizuguchi, H., Xu, Z.L., Sakurai, F., Mayumi, T. & Hayakawa, T. Tight positive regulation of transgene expression by a single adenovirus vector containing the rtTA and tTS expression cassettes in separate genome regions. Hum. Gene Ther. 14, 1265–1277 (2003).

    Article  CAS  Google Scholar 

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  1. Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, United Kingdom

    Arkadiusz Welman, Jane Barraclough & Caroline Dive

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  1. Arkadiusz Welman
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Correspondence to Arkadiusz Welman.

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Welman, A., Barraclough, J. & Dive, C. Generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtTA2S-M2. Nat Protoc 1, 803–811 (2006). https://doi.org/10.1038/nprot.2006.117

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