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.

这是针对计划公告PA-03-107提交的修订后的R21赠款申请，该申请旨在为探索性/开发项目提供申请。该项目的目的是开发一个瞬时遗传转化系统，以用于隐孢子虫。遗传转化是分析基因功能的必不可少的工具，但不能用于孢子虫。加上几乎完整的parvum基因组的序列，需要遗传转化，以通过阐明未知基因的功能并鉴定调节性的功能，将基因组序列转化为生物学和临床相关的信息 序列。我们建议鉴定出在滋养体和早期梅伦特中高度表达的基因，然后将这些基因上游和下游的基因间区域从这些基因中插入含有绿色荧光蛋白报道蛋白的质粒中。这些质粒将开发用于孢子念珠菌的瞬时转化的电穿孔方法。转化后的小孢子将通过对感染细胞培养的荧光显微镜分析直接识别。另外，将使用GFP特异性抗体或反转ccr PCR。与该计划公告的目标一致，该项目是探索性/发展性的，不是假设驱动的。目的是开发与致病微生物研究有关的新技术。 具体目的是： 1。识别在滋养体/早期梅伦特中上调的C parvum基因并构造瞬态 结合调节这些基因表达的区域的转化向量。方法： 在滋养体中高水平表达的rnRNA转录本将通过 定量实时PCR。这些基因两侧的基因间区域将插入GFP表达质粒中。 2。建立一个用于隐孢子虫的瞬态转化系统。 方法：用GFP表达质粒的孢子孢子的电穿孔转化；通过荧光显微镜，抗GFP抗体或反向转移酶PCR检测细胞培养物中转化的人。