Transfection and analysis of transcription in B. malayi

描述（由申请方提供）：丝虫线虫仍然是全球范围内的重大健康问题，超过4000万人患有与这些寄生虫感染相关的疾病。人类丝虫研究的一个主要障碍是缺乏对这些寄生虫进行遗传操作的方法。因此，很少或根本不知道人类丝虫用来调节其基因表达的方法。在上一个项目期间，我们开发了三种不同的方法来瞬时检测B。马来人，人类的子寄生虫。这项技术的发展是我们理解丝虫寄生虫和分子水平的能力的重大进步，并使我们能够对这些生物体中的转录和mRNA加工进行首次详细评估。在本提案中，我们计划在最初供资期间取得的进展的基础上再接再厉。本项目的总体目标是对B中的启动子功能和RNA反式剪接进行详细分析。malayi，使用我们的同源转染系统。我们还将建立在所获得的知识，以开发一个综合的稳定转染方法。为实现这些总体目标，提出了以下具体目标： 1.鉴定与B的HSP70核心启动子中保守转录因子相互作用的元件。马来语。 2.确定HSPT0基因核心启动子的哪些部分在其他B中是保守的。马来人发起人。 3.分析转基因前体mRNA反式剪接所必需的下游顺式作用因子的作用。 4.建立稳定的B细胞转染系统。马来语。

DESCRIPTION (provided by the applicant): Filarial nematodes remain a significant health problem worldwide, with over 40 million individuals suffering from diseases associated with infection of these parasites. A major hindrance in the study of the human filariae has been the lack of methods to genetically manipulate these parasites. As a result, little or nothing is known concerning the methods used by the human filariae to regulate their gene expression. In the previous project period, we developed three different methods to transiently transfect B. malayi, one of the human filial parasites. The development of this technology is a significant advancement in our ability to understand filarial parasites and the molecular level, and has allowed us to make the first detailed assessments of transcription and mRNA processing in these organisms. In this proposal, we plan to build upon the advances made in the initial funding period. The overall goals of this project will be to conduct a detailed analysis of promoter function and RNA trans splicing in B. malayi, using our homologous transfection system. We will also build upon the knowledge gained to develop an integrative stable transfection method. To accomplish these overall goals, the following specific aims are proposed: 1. To identify elements interacting with conserved transcriptional factors in the HSP70 core promoter of B. malayi. 2. To determine which portions of the core promoter of the HSPT0 gene are conserved in other B. malayi promoters. 3. To dissect the role of downstream cis acting factors necessary for trans splicing of transgenic pre-mRNAs. 4. To develop a stable transfection system for B. malayi.