Development of Genetic Tools in Entamoeba histolytica

溶组织内阿米巴遗传工具的开发

• 批准号: 8391038
• 负责人: UPINDER SINGH
• 金额: $ 23.55万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391038
• 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

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. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica is an important pathogen with an impact on human health on a global scale. Despite a wealth of available genomic data, the field suffers from a lack of easy genetic tools to characterize genes and pathways of interest. We are interested in developing novel genetic tools for Entamoeba histolytica, which will advance the study of this parasite and help us better understand the virulence determinants important for disease causation.

描述（由申请人提供）：原生动物寄生虫溶组织内阿米巴引起痢疾和肝脓肿，是全世界寄生虫病最常见的死亡原因之一。一些与发病机制有关的寄生虫遗传决定因素已得到表征；然而，尚未完全了解其致病机制。更彻底地了解疾病的分子基础非常重要，因为 NIAID 将溶组织内阿米巴列为生物恐怖主义关注的 B 类病原体。然而，由于缺乏可靠且简单的遗传工具，进展受到限制。来自基因组计划、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进行遗传分析的基因子集。目前有几种对溶组织内阿米巴进行基因操作的方法，但这些工具有限，而且需要时间和劳动力密集型的性质，因此缺乏大规模使用的便利性，需要高水平的药物选择及其潜在的副作用 效应，以及由于可能缺乏重组而缺乏在 DNA 水平上破坏基因的方法。我们的目标是开发几种新型基因操作工具，重点关注 RNA、蛋白质和 DNA 水平的操作，以应用于溶组织内阿米巴。首先，我们将使用内源性 RNAi 机制和反义小 RNA 优化新型基因沉默。我们将通过开发这种沉默方法来推进该工具：（i）使用基因片段，（ii）多个基因，以及（iii）以受监管的方式。其次，我们的目标是开发一种使用去稳定结构域方法（基于 FK506 结合蛋白和二氢叶酸还原酶）在蛋白质水平上调节表达的工具。我们将开发一种工具，其中以可调节的方式表达蛋白质的显性版本将成为产生功能丧失突变体的策略。第三，我们将建立一个使用piggyBac转座酶将外源DNA整合到基因组中的系统。我们的目标是产生具有随机、单一整合的寄生虫，以便一个基因可以与一种表型相关联。这种方法虽然风险较高，但已在许多不同的细胞类型中取得了成功，值得尝试，因为整合到基因组中将允许采取新的方法（基因破坏、启动子捕获等）。总之，这些新的遗传工具应该提供更复杂的遗传操作方法，并将成为内阿米巴研究界的一大进步。 公共卫生相关性：溶组织内阿米巴是一种重要的病原体，在全球范围内影响人类健康。尽管有大量可用的基因组数据，但该领域仍缺乏简单的遗传工具来表征感兴趣的基因和通路。我们有兴趣开发针对溶组织内阿米巴的新型遗传工具，这将推进对这种寄生虫的研究，并帮助我们更好地了解对疾病致病重要的毒力决定因素。