Genetic Transformation of Cryptosporidium parvum

小隐孢子虫的遗传转化

• 批准号: 6792921
• 负责人: GIOVANNI WIDMER
• 金额: $ 19.81万
• 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 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6792921
• 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.

这是根据计划公告 PA-03-107 提交的修订版 R21 拨款申请，该计划征求探索性/开发项目的申请。该项目的目标是开发隐孢子虫瞬时遗传转化系统。遗传转化是分析基因功能的重要工具，但不适用于小隐孢子虫。连同几乎完整的 C. parvum 基因组序列一起，需要进行遗传转化，通过阐明未知基因的功能并识别调控因子，将基因组序列转化为生物学和临床相关信息。 序列。我们建议鉴定在滋养体和早期分裂体中高度表达的基因，并将这些基因的上游和下游基因间区域插入携带绿色荧光蛋白报告基因的质粒中。将用这些质粒开发用于瞬时转化小隐孢子虫子孢子的电穿孔方法。转化的微小念珠菌将通过感染细胞培养物的荧光显微镜分析直接鉴定。或者，将使用 GFP 特异性抗体或逆转录 PCR。与本计划公告的目标一致，该项目是探索性/开发性的，而不是假设驱动的。目标是开发与病原微生物研究相关的新技术。 具体目标是： 1. 鉴定在滋养体/早期 Meront 中上调的 C parvum 基因并构建瞬态 转化载体包含调节这些基因表达的区域。方法： 在滋养体和早期裂体中高水平表达的 rnRNA 转录物将通过以下方法鉴定 实时定量PCR。这些基因侧翼的基因间区域将被插入 GFP 表达质粒中。 2. 建立小隐孢子虫瞬时转化体系。 方法：用GFP表达质粒电穿孔转化子孢子；通过荧光显微镜、抗 GFP 抗体或逆转录酶 PCR 检测细胞培养物中的转化体。