EGSi, A Tool for Gene Inactivation in Trypanosomatids

描述(由申请人提供)：该项目旨在为两种相关致病原虫布氏锥虫和大利什曼原虫的高通量基因功能研究开发最佳的调节基因失活。RNAi在布氏毛滴虫中效果良好，但在许多情况下并不会使基因表达失活。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核糖核酸酶P介导的基因失活作为评估这两个物种基因功能的工具。该方法利用了RNaseP的有效RNA切割活性，该活性通常在tRNA成熟过程中发挥作用，但切割将通过互补的外部引导序列(EGS)RNA定向到特定的mRNAs。EGS与靶mRNA的相互作用产生类似于前体tRNA的结构，从而被RNaseP特异性切割靶mRNA。EGS将与可调节的启动子一起整合到寄生虫基因组中，以控制其生产，从而控制mRNA的切割。我们建议：(1)开发和评估外引导序列干扰(EGSi)作为布鲁氏锥虫基因沉默的工具；(2)将这一方法应用于布氏锥虫。这一建议旨在开发一种替代和/或互补的方法来以调节的方式和在RNA水平上灭活布氏毛滴虫和主要毛滴虫的基因表达，以绕过由于这些生物是二倍体而引起的并发症。该项目预计将确立这一方法的原则证明，其目的是补充和补充这些生物基因组测序项目迅速出现的基因发现。

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.