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Nat Method2006_A versatile tool for conditional gene expression and knockdown(一种双功能表达系统).pdf

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Nat Method2006_A versatile tool for conditional gene expression and knockdown(一种双功能表达系统)

A versatile tool for conditional gene expression and knockdown Jolanta Szulc1,3, Maciej Wiznerowicz1,2,3, Marc-Olivier Sauvain1,2, Didier Trono1,2,4 Patrick Aebischer1,4 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 promoterdriven transgenes or Pol III promotercontrolled 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. As the genome sequence of a growing number of species become available, there is an increasing need for tools allowing the fast and efficient evaluation of gene function. In particular, systems for conditional transgenesis and gene knockdown are needed that allow for tight control over expression of transgenes and endo- genous genes. Despite major improvements to drug-inducible systems designed for this purpose, several key issues remain to be addressed. The systems developed so far rely on the transactivation of artificial promoters by chimeric transcription factors2,3. Although this most often results in high-level transgene expression, the use of native cellular promoters or enhancer elements would