Reverse genetics using RNAi in non-mouse mammals

描述（由申请人提供）： 小鼠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。它已被用来揭示基因功能的正常以及在体内致病途径，并产生疾病模型，包括许多年龄依赖性疾病。然而，基因敲除技术的复杂性、耗时长和成本高限制了其广泛应用。此外，该技术仅限于在小鼠中进行研究，而在其他哺乳动物物种中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具有序列同源性的短RNA双链体（称为小干扰RNA或siRNA）或由Pol III或Pol II启动子合成的短发夹RNA（shRNA）来介导序列选择性抑制多种真核生物中的基因表达。我们最近的实验表明，Pol II表达的shRNA可以沉默靶基因的表达，在转基因小鼠中观察到敲除表型。基于我们在小鼠中的成功，我们建议以大鼠为模型，研究该技术在其他哺乳动物物种中用于反向遗传学的潜力。我们的RNAi策略使用Pol II合成的shRNA。我们的目标是建立一个概念，即反向遗传学可以使用RNAi在哺乳动物物种而不是小鼠。我们建议（1）基于慢病毒递送技术制备并测试针对大鼠sod2基因的转基因构建体，以及（2）产生并分析转基因大鼠，并确定RNAi策略是否可以敲除大鼠sod2基因并产生预期的sod2基因缺陷的表型，如在小鼠中观察到的。如果成功，这项工作将产生一种新的氧化应激和衰老大鼠模型，重要的是，建立RNAi技术可用于研究基因功能并在大鼠和非小鼠哺乳动物物种中创建疾病模型的概念。

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.