Genetic Transformation of Cryptosporidium parvum

小隐孢子虫的遗传转化

• 批准号: 6888520
• 负责人: GIOVANNI WIDMER
• 金额: $ 23.78万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6888520
• 关键词: Cryptosporidium; RNA directed DNA polymerase; antiantibody; biotechnology; electroporation; fluorescence microscopy; gene delivery system; gene expression; genetic manipulation; genetic mapping; genetic regulation; green fluorescent proteins; laboratory mouse; messenger RNA; method development; microorganism growth; plasmids; polymerase chain reaction; protozoal genetics; spores; tissue /cell culture; transfection /expression vector

This is a revised R21 grant application submitted in response to Program Announcement PA-03-107 which solicits applications for exploratory/developmental projects. The goal of this project is to develop a transient genetic transformation system for Cryptosporidiumparvum. Genetic transformation is an essential tool for analyzing gene function, but is not available for C. parvum. Together with the almost completed sequence of the C. parvum genome, genetic transformation is needed to translate genomic sequence into biologically and clinically relevant information by elucidating the function of unknown genes and identifying regulatory sequences. We propose to identify genes which are highly expressed in trophozoites and early meronts and insert upstream and downstream intergenic regions from these genes into plasmids carrying the green fluorescent protein reporter. Electroporation methods for the transient transformation of C. parvum sporozoites will be developed with these plasmids. Transformed C. parvum will be identified directly by fluorescent microscopical analysis of infected cell cultures. Alternatively, GFP specific antibodies or reversetranscription PCR will be used. Consistent with the goals of this Program Announcement, this project is exploratory/developmental and is not hypothesis-driven. The goal is the development of a new technique relevant to the study of a pathogenic microorganism. The specific aims are: 1. Identify C parvum genes upregulated in trophozoites/early meronts and construct transient transformation vectors incorporating regions regulating the expression of these genes. Approach: rnRNA transcripts expressed at high level in trophozoites and early meronts will be identified by quantitative real-time PCR. Intergenic regions flanking these genes will be inserted into GFP expression plasmids. 2. Establish a transient transformation system for Cryptosporidium parvum. Approach: Transformation by electroporation of sporozoites with GFP expression plasmids; detection of transformants in cell culture by fluorescent microscopy, anti-GFP antibodies, or reverse-transcriptase PCR.

这是为响应项目公告PA-03-107而提交的修订后的R21拨款申请，该公告征求探索性/开发性项目的申请。本课题的目的是建立隐孢子虫的瞬时遗传转化系统。遗传转化是分析基因功能的重要工具，但并不适用于小孢子虫。再加上已基本完成的小孢子虫基因组序列，通过阐明未知基因的功能和鉴定调控基因，需要进行遗传转化，将基因组序列转化为生物学和临床相关信息