RNA Interference as a Metabolic Engineering Tool

RNA 干扰作为代谢工程工具

• 批准号: 6844927
• 负责人: WILLIAM E. BENTLEY
• 金额: $ 18.04万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6844927
• 关键词: N glycosidase; RNA interference; bioengineering /biomedical engineering; biotechnology; cell cycle; cell line; cell proliferation; double stranded RNA; fluorescent in situ hybridization; fusion gene; gene expression; gene induction /repression; glycoprotein structure; glycosylation; microarray technology; northern blottings; polymerase chain reaction; protein engineering; recombinant proteins; synthetic nucleic acid; transfection /expression vector; western blottings

DESCRIPTION (provided by applicant): We have begun to explore controllable in vivo strategies that attempt to manipulate host function with the specific intention of altering the biosynthetic landscape in favor of recombinant protein synthesis. In this proposal, RNA interference (RNAi) or post-translational gene silencing, is explored as a metabolic engineering controller that downregulates targeted host gene expression for this purpose. To date, there have been no related reports in any expression system. We expect that this approach will complement the already proven strategies in metabolic engineering of complementation, mutation, deletion, and allele substitution. The RNAi segment length, target sequences, timing, and method of administration are all subjects of our investigation. Also, we will address two key aspects of protein expression: (1) product yield and (2) product quality. In both cases, preliminary evidence suggests that an RNA-specific gene silencing approach will work. In the first case, we will target the eukaryotic cell cycle. In the second case we will target N-glycosylation of recombinant proteins produced in insect systems. The selection of insect systems for the proposed RNAi work is predicated on several factors: (1) there exists a significant body of knowledge pertaining to insect genetics, including cell cycle circuitry and regulation, (2) much of the pioneering RNAi work has been accomplished in insects (e.g., Drosophila), (3) our laboratory has developed a track record for exploiting insect cells and insect larvae, and (4) DNA microarrays are available that will enable more comprehensive understanding of the mechanisms and efficacy of the RNAi-based metabolic engineering. Intellectual Merit of the Proposed Activity - The development of RNAi as a sensitive and specific controller will open new avenues for metabolic engineering of cells, tissues, and animals. Namely, the downregulation of specific genes, including those in complex pathways, complements existing approaches based on augmenting genotype. Importantly, RNAi is known to elicit minimal side effects or pleiotropy when it is processed properly.

描述（由申请人提供）：我们已经开始探索可控的体内策略，其试图操纵宿主功能，具体目的是改变生物合成景观以有利于重组蛋白合成。在这个提议中，RNA干扰（RNAi）或翻译后基因沉默，被探索作为一个代谢工程控制器，下调靶向宿主基因表达为此目的。迄今为止，在任何表达系统中都没有相关报道。 我们希望这种方法将补充已经证明的策略，在代谢工程的互补，突变，删除和等位基因取代。RNAi片段长度、靶序列、时机和给药方法都是我们研究的主题。此外，我们将解决蛋白质表达的两个关键方面：（1）产品产量和（2）产品质量。在这两种情况下，初步证据表明RNA特异性基因沉默方法将起作用。在第一种情况下，我们将靶向真核细胞周期。在第二种情况下，我们将针对昆虫系统中产生的重组蛋白的N-糖基化。用于所提出的RNAi工作的昆虫系统的选择基于几个因素：（1）存在与昆虫遗传学有关的大量知识，包括细胞周期电路和调控，（2）许多开创性的RNAi工作已经在昆虫中完成（例如，果蝇），（3）我们的实验室已经开发了利用昆虫细胞和昆虫幼虫的跟踪记录，（4）DNA微阵列是可用的，这将使人们能够更全面地了解基于RNAi的代谢工程的机制和功效。 拟议活动的智力价值-RNAi作为一种敏感和特异性控制器的发展将为细胞、组织和动物的代谢工程开辟新的途径。也就是说，下调特定的基因，包括那些在复杂的途径，补充现有的方法的基础上增加基因型。重要的是，已知RNAi在适当处理时引起最小的副作用或多效性。