EGSi, A Tool for Gene Inactivation in Trypanosomatids

EGSi，锥虫基因失活工具

• 批准号: 6760173
• 负责人: REZA Salavati SALAVATI
• 金额: $ 9.55万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6760173
• 关键词: DNA directed RNA polymerase; Leishmania major; RNA; Trypanosoma; Trypanosoma brucei rhodesiense; cell line; gene expression; genetic regulation; messenger RNA; method development; polymerase chain reaction; protozoal genetics; ribonuclease P

DESCRIPTION (provided by the applicant): This project is designed to develop optimal regulated gene inactivation for high throughput gene function studies in the two related pathogenic protozoa Trypanosoma brucei and Leishmania major. RNAi works well in T. brucei but does not inactivate gene expression in many cases. In addition, many attempts in a number of labs have been unable to achieve RNAi-mediated gene inactivation in Leishmania. We propose to develop endogenous cellular RNase P-mediated gene inactivation as a tool for assessing gene function in these two species. The approach exploits the efficient RNA cleavage activity of RNase P, which normally functions in tRNA maturation, but cleavage will be directed to specific mRNAs by a complementary external guide sequence (EGS) RNA. The interaction of the EGS with the target mRNA results in a structure resembling a precursor tRNA and consequently specific cleavage of target mRNA by RNase P. The EGS will be integrated into the parasite genome along with a regulatable promoter in order to control its production and hence mRNA cleavage. We propose to: (1) develop and assess external guide sequence interference (EGSi) as a tool for gene silencing in T. brucei and (2) adapt this approach to L. major. This proposal is designed to develop an alternative and/or complementary means to inactivate gene expression in T. brucei and L. major in a regulated fashion and at the RNA level to bypass complications due to these organisms being diploid. This project is expected to establish the proof of principal of this approach and it is designed to complement and supplement the gene discovery that is rapidly emerging from the Genome Sequencing Projects on these organisms.

描述（由申请人提供）：本项目旨在开发两种相关致病性原生动物布氏锥虫和硕大利什曼原虫中高通量基因功能研究的最佳调控基因失活。RNAi在T.但在许多情况下不抑制基因表达。此外，许多实验室的许多尝试都无法在利什曼原虫中实现RNAi介导的基因失活。我们建议开发内源性细胞RNase P介导的基因失活作为评估这两个物种的基因功能的工具。该方法利用了RNase P的有效RNA切割活性，其通常在tRNA成熟中起作用，但切割将通过互补外部指导序列（EGS）RNA引导至特定mRNA。EGS与靶mRNA的相互作用导致类似于前体tRNA的结构，并因此导致靶mRNA被RNA酶P特异性切割。EGS将与可调节的启动子一起沿着整合到寄生虫基因组中，以控制其产生并因此控制mRNA切割。本研究的主要目的是：（1）开发和评价外部指导序列干扰（EGSi）作为T.（2）将该方法应用于L.少校该建议旨在开发一种替代和/或互补的方法来在T. brucei和L.主要是以调节的方式和在RNA水平上绕过由于这些生物体是二倍体而引起的并发症。该项目预计将建立这种方法的主要证据，它的目的是补充和补充基因发现，这是迅速出现的基因组测序项目对这些生物体。