Development of Genetic Tools in Entamoeba histolytica

溶组织内阿米巴遗传工具的开发

基本信息

批准号：
8515932
负责人：
UPINDER SINGH
金额：
$ 18.45万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8515932
关键词：
Adverse effects Bioterrorism Cause of Death Communities DNA DNA Integration Data Development Dihydrofolate Reductase Disadvantaged Disease Dominant-Negative Mutation Dysentery Entamoeba Entamoeba histolytica Essential Genes Etiology Exploratory/Developmental Grant Gene Expression Gene Silencing Genes Genetic Genetic Determinism Genetic Recombination Genome Genomics Grant Health Human Insecta Liver Abscess Maintenance Methods Molecular Mutagenesis National Institute of Allergy and Infectious Disease Nature Parasites Parasitic Diseases Pathogenesis Pathway interactions Pharmaceutical Preparations Phenotype Plasmids Plasmodium Proteins Proteomics RNA RNA Interference Research Small RNA System Tacrolimus Binding Proteins Time Transgenic Organisms Transposase Virulence Work base cell type embryonic stem cell experience gene replacement genetic analysis genetic manipulation high risk interest loss of function mutant novel novel strategies pathogen promoter research study success tool

项目摘要

DESCRIPTION (provided by applicant): The protozoan parasite Entamoeba histolytica causes dysentery and liver abscesses and is one of the most common causes of death from parasitic disease worldwide. Some parasite genetic determinants involved in pathogenesis have been characterized; however, a complete understanding of the pathogenic mechanisms has not been achieved. A more thorough understanding of the molecular basis of diseases is important as E. histolytica is classified by NIAID as a class B agent of concern for bioterrorism. However, progress has been limited by the paucity of reliable and easy genetic tools. Data from genome projects, microarray experiments and proteomic studies have identified subsets of genes that can be prioritized for genetic analysis. Several methods to genetically manipulate E. histolytica are available but the tools are limited and suffer from time and labor-intensive nature and thus lack of ease for large-scale use, the need for high levels of drug selection and its potential side effects, and the lack of methods to disrupt genes on the DNA level due to potential lack of recombination. We aim to develop several novel genetic manipulation tools, which focus on manipulation at the RNA, protein, and DNA levels for application in E. histolytica. First, we will optimize a novel gene silencing using the endogenous RNAi machinery and antisense small RNAs. We will advance this tool by developing this approach to silence (i) using gene fragments, (ii) multiple genes, and (iii) in a regulated manner. Second, we aim to develop a tool to regulate expression on the protein level using a destabilization domain approach (based on the FK506 binding protein and dihydrofolate reductase). We will develop a tool where expression of dominant version of a protein in a regulatable fashion will be a strategy to generate a loss- of function mutant. Third, we will establish a system to integrate foreign DNA into the genome using piggyBac transposase. We aim to generate parasites with random, single integration, so that one gene can be associated to one phenotype. This approach, although high-risk, has succeeded in many different cell types and is worth attempting because integration into the genome will allow novel approaches to be undertaken (gene disruption, promoter trap, etc). In conclusion, these new genetic tools should provide more sophisticated methods of genetic manipulation and will be a great advance for the Entamoeba research community.

描述（由申请人提供）：原生动物寄生虫结构症引起痢疾和肝脓肿，是全球寄生虫病死亡的最常见原因之一。已经表征了一些参与发病机理的寄生虫遗传决定因素。但是，尚未完全了解致病机制。对疾病的分子基础的更彻底的理解很重要，因为E. histolictica被NIAID归类为B级B级对生物恐怖主义的关注。但是，由于缺乏可靠和简单的遗传工具，进步受到了限制。来自基因组项目，微阵列实验和蛋白质组学研究的数据已经确定了可以优先考虑遗传分析的基因子集。可以使用几种遗传操纵E. tosolytica的方法，但工具受到限制，并且受到时间和劳动密集型性质的损失，因此缺乏对大规模使用的轻松，需要高水平的药物选择及其潜在的侧面效果以及由于潜在的重组而缺乏在DNA水平上破坏基因的方法。我们旨在开发几种新型的基因操纵工具，这些工具的重点是在RNA，蛋白质和DNA水平上进行操作，以应用于E. histolytica。首先，我们将使用内源性RNAi机械和反义小RNA优化一种新型的基因沉默。我们将使用基因片段，（ii）多个基因和（iii）以调节方式开发这种静音方法来推进此工具。其次，我们旨在使用不稳定域方法开发一种工具来调节蛋白质水平的表达（基于FK506结合蛋白和二氢叶酸还原酶）。我们将开发一种工具，其中以可调节方式的蛋白质的显性版本表达将成为产生功能突变体损失的策略。第三，我们将使用PiggyBac转座酶建立一个将外源DNA整合到基因组中的系统。我们旨在生成具有随机的单一积分的寄生虫，以便一个基因可以与一种表型相关联。这种方法虽然高风险，但在许多不同的细胞类型中都取得了成功，值得尝试，因为整合到基因组中将允许采用新颖的方法（基因破坏，启动子陷阱等）。总之，这些新的遗传工具应提供更复杂的遗传操作方法，对于Entamoeba研究界来说将是一个很好的进步。