A Versatile Tool for Conditional Gene Expression and Knockdown

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. However, currently available systems often exhibit limited in vivo functionality due to leakiness, insufficient levels of induction, lack of tissue specificity or prohibitively complicated designs. To overcome these limitations we have developed a lentivector-based, conditional gene expression system for tetracycline-controllable repression of polymerase II-driven transgenes or polymerase III- controlled sequences encoding small inhibitory hairpin RNAs (shRNAs). Our system allows for tightly controlled, robust, and fully reversible gene expression or knockdown in cell lines, primary human haemopoietic progenitor cells and embryonic mouse stem cells (ES) in vitro, in both Tet-on or Tet-off configurations. Tight and efficient conditional expression of human Glial Cell Line Derived Neurotrophic Factor (GDNF) in the rat striatum and substantia nigra was achieved using this vector offering numerous opportunities for gene therapy approaches, modeling human diseases or studying gene function in central nervous system. When used for conditional knockdown this system allows for robust and fully reversible control of endogenous TP53 tumor supressor in human cancer cells xenotransplanted into nude mice, thus opening promising possibilities for both basic and applied cancer research. Finally, we probed the potential of our system by generating transgenic mice either by infection of fertilized oocytes with LV vectors or blastocyst transfer of transduced ES cells. These results pave the novel ways for basic or translational research, the development of gene-based therapeutics, and for creation of animal models of human diseases. M.W. and D.T. patented part of the technology described in this article.