Accelerating reverse genetics in mice by multi-knockdowns using RNAi

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DESCRIPTION (provided by applicant): Gene knockout in mice is a powerful tool for the study of gene function, at the whole body level. Generation of the knockout mouse models relies on homologous recombination within embryonic stem (ES) cells. This method, although highly effective, is also laborious, time-consuming and very costly. Evidence of dysfunction of two, or more, related genes will help to determine whether the related genes function in parallel, or in a stepwise fashion, within a pathway. Even the generation of a double knockout mouse model, with the knockout approach, will be a time-consuming process. The partial deletion of some the disease-associated genes will model the human diseases in animals, and thusly, will accelerate the study of the disease mechanisms and associated therapies. However, the traditional knockout proves to be difficult, as it will require performance of a laborious procedure to knock in a mutant gene, into the same genomic location. RNA interference (RNAi) can mediate sequence-specific and dose-dependent destruction of complementary RNA molecules, such as mRNA, leading to the dysfunction of the targeted gene. Sustained RNAi can be achieved by the introduction of a gene-based short hairpin RNA (shRNA) expression cassette. Despite early optimism that RNAi transgenic mice could be generated readily and quickly, the success in knocking down endogenous genes, which recapitulates phenotypes of the knockouts, has only been demonstrated in embryos, and has not been reported in all the tissues of postnatal transgenic mice. I have generated a potent shRNA system for expressing shRNA in vivo. Here, I propose to explore whether RNAi can serve as an alternative to gene knockout technology, in the generation of animal models that are depleted of two, or more, related genes. Once established, this multi-RNAi knockdown technology can be widely used to quickly probe the roles of multiple related genes. Combined with the inducible gene inhibition, this method will provide an unprecedented ease in the manipulation of gene expression levels, in mammals. With this useful tool, the investigation of gene function, as well as human diseases, will be dramatically accelerated.