Reverse genetics using RNAi in non-mouse mammals

描述(由申请人提供)： 小鼠的基因敲除技术在研究哺乳动物的基因功能方面发挥了重要作用。它已被用来揭示正常和体内致病途径中的基因功能，并用于建立包括许多年龄相关性疾病在内的疾病模型。然而，基因敲除技术的复杂性、时间的长短和高昂的成本限制了它的广泛应用。此外，这项技术仅限于在小鼠身上进行研究，其他哺乳动物物种的基因敲除只能通过克隆来完成，这在技术上比通过同源重组进行基因敲除更加复杂、困难和昂贵。RNA干扰(RNAi)可以通过引入与靶基因序列同源的短RNA双链(称为小干扰RNAs或siRNAs)或从Pol III或Pol II启动子合成的短发夹状RNAs(ShRNAs)来介导对多种真核生物中基因表达的序列选择性抑制。我们最近的实验证明，Pol II表达的shRNA可以沉默靶基因的表达，在一定程度上可以在转基因小鼠中观察到敲除表型。基于我们在小鼠身上的成功，我们建议以大鼠为模型，研究这项技术在其他哺乳动物物种反向遗传学中的应用潜力。我们的RNAi策略使用Pol II合成的shRNA。我们的目标是建立这样一个概念，即可以在小鼠以外的哺乳动物物种中使用RNAi进行反向遗传学。我们建议(1)基于慢病毒传递技术制作并测试针对大鼠Sod2基因的转基因构建；(2)制备并分析转基因大鼠，并确定RNAi策略是否能够敲除大鼠Sod2基因并产生预期的Sod2基因缺失的表型，如在小鼠中观察到的那样。如果成功，这项工作将产生一种新的氧化应激和衰老的大鼠模型，重要的是，建立了RNAi技术可以用于研究基因功能并在大鼠和非小鼠哺乳动物物种中建立疾病模型的概念。

DESCRIPTION (provided by applicant): Gene knockout technology in mice has been instrumental in investigating gene functions in mammals. It has been used to reveal gene functions in normal as well as in pathogenic pathways in vivo, and to generate disease models including many age-dependent diseases. However, the technical complexity, the length of time and the high cost of gene knockout approach has limited its wider use. Additionally, the technology is limited to investigations in mice and gene knockouts in other mammalian species can only be done with cloning, which is technically even more complex, difficulty and expensive than gene knockout by homologous recombination. RNA interference (RNAi) can mediate sequence-selective suppression of gene expression in a wide variety of eukaryotes by introducing short RNA duplexes (called small interfering RNAs or siRNAs) with sequence homologies to the target gene or short hairpin RNAs (shRNAs) synthesized from a Pol III or Pol II promoters. Our recent experiments have demonstrated that shRNA expressed by Pol II can silence target gene expression to the extent that knockout phenotypes are observed in transgenic mice. Based on our success in mice, we propose to investigate the potential for this technology to be used in reverse genetics in other mammalian species, using rat as a model. Our RNAi strategy uses Pol II synthesized shRNA. Our goal is to establish the concept that the reverse genetics can be done using RNAi in mammalian species other than the mouse. We propose to (1) make and test the transgene construct against the rat sod2 gene based on lentivirus delivery technology and (2) generate and analyze the transgenic rat and determine whether the RNAi strategy can knock down the rat sod2 gene and produce phenotypes expected of sod2 gene deficiency, as observed in mice. If successful, this work will produce a new rat model in oxidative stress and aging, and importantly, establish the concept that RNAi technology can be used to investigate gene functions and create disease models in rat and non-mouse mammalian species.