Genetic Transformation of Cryptosporidium parvum

这是一份修订后的R21赠款申请，是为了响应项目公告PA-03-107而提交的，该公告征集探索/开发项目的申请。本研究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，但对C.小的结合C.在微小基因组中，需要遗传转化来通过阐明未知基因的功能和鉴定调控基因来将基因组序列翻译成生物学和临床相关信息 序列的我们建议确定在滋养体和早期裂殖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。采用电穿孔法对C.将用这些质粒产生小孢子。变形C通过对感染的细胞培养物进行荧光显微镜分析来直接鉴定细小病毒。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，本项目是探索性/发展性的，不是假设驱动的。目标是开发一种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/早期裂殖体中上调的微小念珠菌基因并构建瞬时 整合了调节这些基因表达的区域的转化载体。方法： 在滋养体和早期裂殖体中高水平表达的mRNA转录本将通过以下方法鉴定： 实时定量PCR。将这些基因侧翼的基因间区域插入GFP表达质粒中。 2.微小隐孢子虫瞬时转化体系的建立。 方法：用GFP表达质粒通过子孢子电穿孔进行转化;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。

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.