Generation and validation of a novel inducible overexpression library for genome-scale genetic screens in Leishmania

用于利什曼原虫基因组规模遗传筛选的新型诱导过表达文库的生成和验证

• 批准号: 10818854
• 负责人: PHILLIP A YATES
• 金额: $ 3.97万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-13 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10818854
• 关键词: Biology; Cells; Communities; Disease; Drug resistance; Expression Library; Generations; Genetic; Genetic Screening; Genomic Library; Glean; Goals; Growth; Leishmania; Leishmania donovani; Libraries; Life Cycle Stages; Molecular Genetics; Open Reading Frames; Parasites; Pathway interactions; Persons; Pharmacotherapy; Plasmids; Proteins; RNA Interference; Resistance; Resources; Transfection; Trypanosoma brucei brucei; Validation; Visceral; Visceral Leishmaniasis; drug action; expression vector; fitness; genome-wide; human disease; innovation; knock-down; neglected tropical diseases; novel; novel therapeutics; overexpression; protein expression; protein function; resistance gene; tool; vector

Project Summary Leishmania parasites cause a suite of devastating Neglected Tropical Diseases that afflict as many as one million people per year. At least 20,000 are killed each year by the deadly visceral form of the disease, which is caused by L. donovani. Genetic tools for understanding important questions in Leishmania biology are currently quite limited. Most Leishmania species lack the capacity for RNA interference (RNAi), which precludes the implementation of genome-scale RNAi library screens like those that have revolutionized molecular genetics in the related kinetoplastid T. brucei. As is the case for knocking down protein expression via RNAi, expressing proteins at higher levels than normal (i.e., overexpression) can confer resistance to drugs or otherwise enhance survival (Gain-of-Fitness = GnFt), or cause deleterious effects on cell fitness (Loss-of- Fitness = LsFt) that can reveal important pathways and protein functions. Genome-scale overexpression libraries have proven valuable for a wide range of genetic screens in multiple species, including T. brucei. The goal of this proposal is to generate and validate a novel, genome-scale, inducible overexpression library in Leishmania that will serve as a versatile and broadly applicable new genetic tool for the field. We propose to generate a library encoding the majority of L. donovani proteins (~7500 Open Reading Frames, or LdORFs). PCR amplified LdORFs will be directionally cloned into Gateway Entry vectors to facilitate transfer of the library to any Gateway compatible vector (Aim 1), resulting in a LdORFeome plasmid library. The LdORFeome will be transferred into novel inducible Leishmania expression vectors developed in our lab (Aim 1). The resulting library will be stably transfected into Leishmania to generate an inducible Leishmania donovani ORFeome overexpression library (the LdOX library). Because the overall goal is to make the LdOX library available as a resource for the community, we will validate the library by performing proof-of-principle overexpression screens to identify drug resistance genes (GnFt) and pathways required for normal growth (LsFt) (Aim2). The LdOX library will provide an innovative and much-needed new tool for high throughput genetic screens in Leishmania. We anticipate that screens with the LdOX library will have a sustained impact on the field by revealing mechanisms of drug action and resistance, and uncovering proteins and pathways required for surviving any growth condition or lifecycle stage.

项目摘要 利什曼原虫寄生虫引起一套毁灭性的热带疾病，使其遭受与 每年100万人。每年至少有20,000人被这种疾病的致命内脏形式杀死 这是由多诺瓦尼（L. donovani）造成的。利什曼原虫生物学中理解重要问题的遗传工具是 目前非常有限。大多数利什曼原虫物种都缺乏RNA干扰的能力（RNAi）， 阻止了基因组规模的RNAi图书馆屏幕的实施， 相关的动力质体T. brucei中的分子遗传学。与击倒蛋白质表达一样 通过RNAi，以高于正常水平的蛋白质表达蛋白质（即过表达）可以赋予对药物的抗性 或其他提高生存率（拟合获得= GNFT），或对细胞适应性产生有害影响（丧失 - 适应性= LSFT）可以揭示重要的途径和蛋白质功能。基因组规模的过表达 事实证明，图书馆对于包括T. Brucei在内的多种物种中的广泛遗传筛选很有价值。这 该建议的目标是生成和验证一个新颖的基因组规模，可诱导的过表达库 利什曼尼亚（Leishmania）将作为该领域的多功能且广泛适用的新遗传工具。 我们建议生成一个编码大多数多诺瓦尼蛋白的库（〜7500个开放阅读 框架或ldorfs）。 PCR放大的ldorfs将定向到网关进入向量到 促进图书馆转移到任何兼容矢量的任何网关（AIM 1），从而导致LDORFEOME质粒 图书馆。 ldorfeome将被转移到新型的诱导利什曼原虫表达载体中 我们的实验室（目标1）。由此产生的库将稳定转染到利什曼尼亚，以生成诱导的 Leishmania Donovani Orfeome过表达图书馆（LDOX库）。因为总体目标是 使LDOX库作为社区资源可用，我们将通过执行库来验证库 基本过表达筛选，以鉴定耐药性基因（GNFT）和所需的途径 正常生长（LSFT）（AIM2）。 LDOX库将为高通量遗传提供创新且急需的新工具 利什曼尼亚的屏幕。我们预计使用LDOX库的屏幕将对 通过揭示药物作用和耐药性的机制，并发现所需的蛋白质和途径 为了生存任何生长状况或生命周期阶段。