Conditional Gene Expression and Knockdown Using Lentivirus Vectors Encoding shRNA

  • Jolanta Szulc
  • Patrick Aebischer
Part of the Methods in Molecular Biology™ book series (MIMB, volume 434)


Drug-inducible systems allowing the control of transgene expression and knockdown in mammalian cells are invaluable tools for genetic research, and could also play important roles in translational research or gene therapy. We and others have developed a lentivector-based, conditional gene expression system for drug-controllable expression of transgenes and small hairpin RNAs (shRNAs). This system is highly robust and versatile, governing tightly controlled expression of transgenes and endogenous cellular genes (through shRNAs) in various primary and established cell lines in vitro, as well as in vivo in the central nervous system or in human cancer cells xenotransplanted into nude mice. The goal of this article is to provide a concise methodology for construction and manipulation of this conditional lentiviral-based system, and quantitative analyses of drug-inducible transgene expression and gene knockdown both in vitro and in vivo.

Key Words

Conditional gene expression drug-inducible gene knockdown shRNA lentiviral vectors doxycycline 



Authors thank Maciej Wiznerowicz for critical reading of this chapter.


  1. 1.
    Toniatti, C., Bujard, H., Cortese, R., and Ciliberto, G. (2004) Gene therapy progress and prospects: transcription regulatory systems. Gene Ther 11, 649–57.CrossRefPubMedGoogle Scholar
  2. 2.
    Gossen, M., and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89, 5547–51.CrossRefPubMedGoogle Scholar
  3. 3.
    Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–9.CrossRefPubMedGoogle Scholar
  4. 4.
    Margolin, J. F., Friedman, J. R., Meyer, W. K., Vissing, H., Thiesen, H. J., and Rauscher, F. J., 3rd (1994) Kruppel-associated boxes are potent transcriptional repression domains. Proc Natl Acad Sci USA 91, 4509–13.CrossRefPubMedGoogle Scholar
  5. 5.
    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, 669–77.CrossRefPubMedGoogle Scholar
  6. 6.
    Urrutia, R. (2003) KRAB-containing zinc-finger repressor proteins. Genome Biol 4, 231.CrossRefPubMedGoogle Scholar
  7. 7.
    Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243–7.CrossRefPubMedGoogle Scholar
  8. 8.
    Stegmeier, F., Hu, G., Rickles, R. J., Hannon, G. J., and Elledge, S. J. (2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA 102, 13212–7.CrossRefPubMedGoogle Scholar
  9. 9.
    Dickins, R. A., Hemann, M. T., Zilfou, J. T., Simpson, D. R., Ibarra, I., Hannon, G. J., and Lowe, S. W. (2005) Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37, 1289–95.PubMedGoogle Scholar
  10. 10.
    Wiznerowicz, M. and Trono, D. (2005) Harnessing HIV for therapy, basic research and biotechnology. Trends Biotechnol 23, 42–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Szulc, J., Wiznerowicz, M., Sauvain, M. O., Trono, D., and Aebischer, P. (2006) A versatile tool for conditional gene expression and knockdown. Nat Methods 3, 109–16.CrossRefPubMedGoogle Scholar
  12. 12.
    Miyake, K., Flygare, J., Kiefer, T., Utsugisawa, T., Richter, J., Ma, Z., Wiznerowicz, M., Trono, D., and Karlsson, S. (2005) Development of cellular models for ribosomal protein S19 (RPS19)-deficient diamond-blackfan anemia using inducible expression of siRNA against RPS19. Mol Ther 11, 627–37.CrossRefPubMedGoogle Scholar
  13. 13.
    Arrighi, J. F., Pion, M., Wiznerowicz, M., Geijtenbeek, T. B., Garcia, E., Abraham, S., Leuba, F., Dutoit, V., Ducrey-Rundquist, O., van Kooyk, Y., Trono, D., and Piguet, V. (2004) Lentivirus-mediated RNA interference of DC-SIGN expression inhibits human immunodeficiency virus transmission from dendritic cells to T cells. J Virol 78, 10848–55.CrossRefPubMedGoogle Scholar
  14. 14.
    Moffat, J., Grueneberg, D. A., Yang, X., Kim, S. Y., Kloepfer, A. M., Hinkle, G., Piqani, B., Eisenhaure, T. M., Luo, B., Grenier, J. K., Carpenter, A. E., Foo, S. Y., Stewart, S. A., Stockwell, B. R., Hacohen, N., Hahn, W. C., Lander, E. S., Sabatini, D. M., and Root, D. E. (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–98.CrossRefPubMedGoogle Scholar
  15. 15.
    Berns, K., Hijmans, E. M., Mullenders, J., Brummelkamp, T. R., Velds, A., Heimerikx, M., Kerkhoven, R. M., Madiredjo, M., Nijkamp, W., Weigelt, B., Agami, R., Ge, W., Cavet, G., Linsley, P. S., Beijersbergen, R. L., and Bernards, R. (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–7.CrossRefPubMedGoogle Scholar
  16. 16.
    Ngo, V. N., Davis, R. E., Lamy, L., Yu, X., Zhao, H., Lenz, G., Lam, L. T., Dave, S., Yang, L., Powell, J., and Staudt, L. M. (2006) A loss-of-function RNA interference screen for molecular targets in cancer. Nature 441, 106–10.CrossRefPubMedGoogle Scholar
  17. 17.
    Silva, J. M., Li, M. Z., Chang, K., Ge, W., Golding, M. C., Rickles, R. J., Siolas, D., Hu, G., Paddison, P. J., Schlabach, M. R., Sheth, N., Bradshaw, J., Burchard, J., Kulkarni, A., Cavet, G., Sachidanandam, R., McCombie, W. R., Cleary, M. A., Elledge, S. J., and Hannon, G. J. (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37, 1281–8.PubMedGoogle Scholar
  18. 18.
    Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., and Khvorova, A. (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22, 326–30.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Jolanta Szulc
    • 1
  • Patrick Aebischer
    • 1
  1. 1.Neurosciences InstituteSwiss Federal Institute of Technology LausanneSwitzerland

Personalised recommendations