Accelerating reverse genetics in mice by multi-knockdowns using RNAi

描述（由申请人提供）：小鼠基因敲除是在全身水平上研究基因功能的有力工具。敲除小鼠模型的产生依赖于胚胎干细胞内的同源重组。这种方法虽然非常有效，但也很费力、耗时和昂贵。两个或更多相关基因功能障碍的证据将有助于确定相关基因是在一个途径中平行起作用，还是以逐步的方式起作用。即使是用基因敲除方法产生双基因敲除小鼠模型，也将是一个耗时的过程。一些疾病相关基因的部分缺失将在动物中模拟人类疾病，从而加速疾病机制和相关治疗的研究。然而，传统的基因敲除被证明是困难的，因为它需要执行一个费力的程序，将突变基因敲入相同的基因组位置。RNA干扰（RNAi）可以介导互补RNA分子（如mRNA）的序列特异性和剂量依赖性破坏，导致靶基因功能障碍。持续的RNAi可以通过引入基于基因的短发夹RNA （shRNA）表达盒来实现。尽管早期人们乐观地认为RNAi转基因小鼠可以容易而快速地产生，但成功地敲除内源性基因（概括敲除的表型）只在胚胎中得到证实，并没有在出生后转基因小鼠的所有组织中得到报道。我已经生成了一个在体内表达shRNA的有效的shRNA系统。在这里，我建议探索RNAi是否可以作为基因敲除技术的替代品，在产生两个或更多相关基因缺失的动物模型中。一旦建立，这种多rnai敲低技术可以广泛应用于快速探测多个相关基因的作用。结合诱导基因抑制，这种方法将提供前所未有的易于操纵的基因表达水平，在哺乳动物。有了这个有用的工具，对基因功能和人类疾病的研究将大大加快。

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.