Inducible gene silencing in transgenics: RNAi

转基因中的诱导基因沉默：RNAi

• 批准号: 6665029
• 负责人: OLGA A CABELLO
• 金额: $ 15.05万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6665029
• 关键词: biotechnology; functional /structural genomics; gene expression; gene induction /repression; genetic manipulation; genetic techniques; genetic transcription; genetically modified animals; laboratory mouse; messenger RNA; technology /technique development

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to develop protocols for inducible and reversible tissue-specific gene silencing in the mouse. We propose to test the feasibility of an RNA interference (RNAi)- based strategy for gene inactivation in transgenic mice. RNAi is an experimental strategy that takes advantage of a conserved endogenous pathway for gene silencing. This pathway appears to serve a dual function: to guard cells against molecular parasites, and to regulate the chronology of development. RNAi is triggered by double-_stranded RNAs (dsRNA), corresponding to sense and antisense sequences of target genes. The dsRNA precursors are cleaved by Dicer RNAse into short interfering RNAs (siRNAs) that guide an RNA Induced Silencing Complex (RISC) to the target transcript. In a related mechanism, Dicer also cleaves dsRNA precursors to generate _short t emporal RNAs (stRNAs) that act by interfering with translation. RNAi is the most powerful technique available for the functional analysis of C.elegans and Drosophila genomes and systematic targeted inactivation projects are under way. The establishment of RNAi-based gene inactivation technologies in the mouse would have significant advantages over current protocols for gene inactivation. Because the gene silencing mechanism leaves the genomic locus intact, the silencing effect is potentially reversible. In addition, transgenic RNAi would be faster and significantly less costly than current locus inactivation protocols. Recent tissue culture studies provide strong evidence that the fundamental mechanisms for RNAi are evolutionarily conserved and operational in mammalian cells. However, the existence of the interferon response in mammalian cells is an obstacle to the implementation of dsRNA-based strategies. We intend to test four transgenic strategies in mice, each designed to avoid the activation of an interferon response. Our Specific Aims are: 1) To test whether gene silencing in transgenic mice can be accomplished by expression of short palindromic RNAs, which we term "snap-back" RNAs, 2) To test whether gene silencing can be accomplished by expression of long interrupted RNAs, which we term "bubble hybrid" RNAs, 3) To test whether gene silencing in the mouse can be facilitated by co-expression of Dicer RNAse and an RNAi transgene, and 4) To test whether "stRNA"-mediated inhibition of translation is feasible in transgenic mice. We will use two model systems to evaluate the efficacy of gene inactivation: the inhibition of tryrosinase expression in melanocytes and in the Retinal Pigmented Epithelium (RPE), predicting changes in pigmentation, and the inactivation of Rb in lens fiber cells resulting in cataracts and microphthalmia. Once established, RNAi technology will be more flexible than traditional gene knock-out techniques and will greatly accelerate the functional characterization of the mammalian genome.

描述（由申请人提供）：该提案的长期目标是开发小鼠中可诱导和可逆的组织特异性基因沉默的方案。我们建议测试基于 RNA 干扰 (RNAi) 的转基因小鼠基因失活策略的可行性。 RNAi 是一种利用保守的内源途径进行基因沉默的实验策略。该途径似乎具有双重功能：保护细胞免受分子寄生虫的侵害，并调节发育的时间顺序。 RNAi 由双链 RNA (dsRNA) 触发，对应于靶基因的有义和反义序列。 dsRNA 前体被 Dicer RNAse 切割成短干扰 RNA (siRNA)，引导 RNA 诱导沉默复合物 (RISC) 到达目标转录物。在相关机制中，Dicer 还会裂解 dsRNA 前体，生成短时间 RNA (stRNA)，通过干扰翻译发挥作用。 RNAi 是可用于线虫和果蝇基因组功能分析的最强大技术，系统性靶向灭活项目正在进行中。在小鼠中建立基于 RNAi 的基因失活技术将比现有的基因失活方案具有显着的优势。由于基因沉默机制使基因组位点保持完整，因此沉默效应可能是可逆的。此外，转基因 RNAi 比目前的基因座失活方案更快且成本显着降低。最近的组织培养研究提供了强有力的证据，证明 RNAi 的基本机制在进化上是保守的并且在哺乳动物细胞中发挥作用。然而，哺乳动物细胞中干扰素反应的存在是实施基于 dsRNA 的策略的障碍。我们打算在小鼠中测试四种转基因策略，每种策略都旨在避免激活干扰素反应。我们的具体目标是：1) 测试转基因小鼠中的基因沉默是否可以通过表达短回文 RNA（我们称之为“弹回”RNA）来实现，2) 测试是否可以通过表达长中断 RNA（我们称之为“气泡杂合”RNA）来实现基因沉默，3) 测试是否可以通过共表达 Dicer RNAse 和 RNAi 转基因，以及 4) 测试“stRNA”介导的翻译抑制在转基因小鼠中是否可行。我们将使用两个模型系统来评估基因失活的功效：抑制黑色素细胞和视网膜色素上皮 (RPE) 中的酪氨酸酶表达，预测色素沉着的变化，以及导致白内障和小眼症的晶状体纤维细胞中 Rb 失活。一旦建立，RNAi技术将比传统的基因敲除技术更加灵活，并将大大加速哺乳动物基因组的功能表征。