权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

基本信息

批准号：
10666941
负责人：
PHILLIP A YATES
金额：
$ 23.81万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-13 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10666941
关键词：
Acceleration Bar Codes Biology CRISPR/Cas technology Cells Communities Complex DL-alpha-Difluoromethylornithine Defect Disease Drug Targeting Drug resistance Ensure Expression Library Gene Expression Regulation Generations Genes Genetic Genetic Screening Genetic Techniques Genomic Library Glean Goals Growth Hyperactivity Incidence Individual Laboratories Leishmania Leishmania donovani Libraries Life Cycle Stages Melarsoprol Methods Molecular Genetics Open Reading Frames Ornithine Decarboxylase Ornithine Decarboxylase Inhibitor Parasites Pathway interactions Persons Pharmacotherapy Phenotype Plasmids Proteins RNA Interference RNA interference screen Reproducibility Resistance Resources Ribosomal RNA Signal Transduction Stress System Time Transfection Trypanosoma brucei brucei Trypanosoma cruzi Validation Visceral Visceral Leishmaniasis Yeasts dosage drug action experience experimental study expression vector fitness genetic regulatory protein genome-wide human disease inducible gene expression innovation knock-down loss of function neglected tropical diseases new therapeutic target novel novel therapeutics overexpression protein expression protein function resistance gene resistance mechanism tool vector

项目摘要

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.

项目总结