Accelerating reverse genetics in mice by multi-knockdowns using RNAi

描述(申请人提供)：小鼠的基因敲除是在全身水平上研究基因功能的有力工具。基因敲除小鼠模型的产生依赖于胚胎干细胞内的同源重组。这种方法虽然很有效，但也很费力、耗时和成本很高。两个或更多相关基因功能失调的证据将有助于确定相关基因在一条途径内是平行发挥作用，还是以逐步方式发挥作用。即使是用基因敲除的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，从而将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繁琐的程序来将突变基因敲入相同的基因组位置。RNA干扰(RNAi)可以介导序列特异性和剂量依赖性的互补RNA分子的破坏，导致靶基因的功能障碍。持续的RNAi可以通过引入基于基因的短发夹状RNA(ShRNA)表达盒来实现。尽管早期乐观地认为RNAi转基因小鼠可以容易而快速地产生，但成功击倒内源性基因(概括了敲除基因表型的基因)只在胚胎中得到证实，并未在出生后转基因小鼠的所有组织中得到报道。我已经建立了一个有效的shRNA系统来在体内表达shRNA。在这里，我建议探索RNAi是否可以作为基因敲除技术的替代，在缺乏两个或更多相关基因的动物模型的生成中。一旦建立，这种多RNAi敲除技术可以广泛用于快速探索多个相关基因的作用。与可诱导的基因抑制相结合，这种方法将为哺乳动物基因表达水平的操纵提供前所未有的轻松。有了这个有用的工具，基因功能的研究以及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.