Abstract
Drug-inducible systems allowing the control of gene expression in mammalian cells are invaluable tools for genetic research, and could also fulfill essential roles in gene- and cell-based therapy. Currently available systems, however, often have limited in vivo functionality because of leakiness, insufficient levels of induction, lack of tissue specificity or prohibitively complicated designs. Here we describe a lentiviral vector–based, conditional gene expression system for drug-controllable expression of polymerase (Pol) II promoter−driven transgenes or Pol III promoter−controlled sequences encoding small inhibitory hairpin RNAs (shRNAs). This system has great robustness and versatility, governing tightly controlled gene expression in cell lines, in embryonic or hematopoietic stem cells, in human tumors xenotransplanted into nude mice, in the brain of rats injected intraparenchymally with the vector, and in transgenic mice generated by infection of fertilized oocytes. These results open up promising perspectives for basic or translational research and for the development of gene-based therapeutics.
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Acknowledgements
We thank V. Padrun, F. Pidoux, C. Sadeghi, L. Winkel, A. Maillard and P. Colin for excellent technical assistance; A. Quazzola for generating the transgenic mice and S. Liao for genotyping them; I. Malanchi and J. Huelsken for help with nude mice experiments; T. Matthes for help with screening for GATA1 shRNAs; and F. Spitz for the gift of D3 mES cells. This work was supported by grants from the Swiss National Science Foundation (to P.A. and D.T.), the European Union (CONSERT integrated project, D.T.; APOPIS contract, P.A.), the Institut Clayton de la Recherche Geneva, and the National Center for Competence in Research Frontiers in Genetics (D.T.), and the Michael J. Fox Foundation (P.A.).
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M.W. and D.T. patented part of the technology described in this article.
Supplementary information
Supplementary Fig. 1
In vitro drug-controllable transgene expression. (PDF 62 kb)
Supplementary Fig. 2
Reversibility of extended tTRKRAB repression. (PDF 119 kb)
Supplementary Fig. 3
Quantification of conditional cycling of p53 knockdown and GFP expression in vitro. (PDF 188 kb)
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Szulc, J., Wiznerowicz, M., Sauvain, MO. et al. A versatile tool for conditional gene expression and knockdown. Nat Methods 3, 109–116 (2006). https://doi.org/10.1038/nmeth846
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DOI: https://doi.org/10.1038/nmeth846
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