Cryptococcus knockout and tag resource

隐球菌敲除和标签资源

基本信息

批准号：
10159073
负责人：
Hiten D Madhani
金额：
$ 61.9万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
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：
2012-05-01 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10159073
关键词：
Acquired Immunodeficiency Syndrome Animal Model Behavior Biological Biology Cell Wall Cessation of life Clinical Code Collection Communities Coupled Cryptococcus Cryptococcus neoformans Deposition Diagnosis Encapsulated Epitopes Foundations Fungal Meningitis Gene Deletion Gene Proteins Generations Genes Genetic Recombination Genome Genomics Goals Grant Hand Haploidy Human Human Resources Hypoxia Individual Infection Informatics Infrastructure Iron Knock-out Knowledge Letters Life Lung infections Medical Melanins Meningoencephalitis Modeling Mus Mycoses Oligonucleotides Oxidative Stress Patients Pharmaceutical Preparations Phase Phenotype Polysaccharides Process Production Property Proteins Proteome Quality Control Reagent Research Resistance Resources Saccharomyces cerevisiae Saccharomycetales Southern Blotting Stress System The science of Mycology Training Urease Virulence Virulence Factors Work Yeast Model System base capsule deletion library design experimental study fitness fungal genetics gene gun genome resource genome-wide homologous recombination human pathogen in vitro Assay in vivo informatics infrastructure interest knockout gene mortality mutant nitrosative stress novel therapeutics pathogen pathogenic fungus protein function quorum sensing recruit tool trait whole genome

项目摘要

The effective diagnosis and treatment of life-threatening fungal infections in humans is a major unmet clinical challenge. Well-annotated genomic sequences of the major human fungal pathogens have been available for a number of years, creating an opportunity to revolutionize medical mycology through the application of systematic genome-wide approaches. However, to be maximally useful, whole-genome sequences need to be coupled with the genome-wide biological resources. The availability of genome-wide knockout and tagged gene collections has been instrumental in dissecting the fundamental biology of the model yeast Saccharomyces cerevisiae. Our premise is that the generation of analogous collections in pathogenic fungi would create powerful experimental tools enabling comprehensive approaches to elucidating the basis of fungal virulence in the mammalian host and to develop drugs. Such tools accelerate research of the community through the application of existing in vivo and in vitro assays to functionally profile the behavior of all genes and proteins in any process of interest. Cryptococcus neoformans is an encapsulated budding yeast that is the most common cause of fungal meningitis. An estimated ~1,000,000 cases result in ~600,000 deaths each year. One-third of deaths in AIDS patients worldwide are the result of Cryptococcal meningoencephalitis. This important fungal pathogen has haploid genetics and homologous recombination, making it an excellent model pathogen and model organism. There are 6967 predicted coding sequences. In the initial period of this grant, we developed an informatics infrastructure, designed and purchased ~70,000 oligonucleotides, recruited/trained personnel and successfully constructed and deposited 4042 gene knockout strains using biolistic (“gene gun”) transformation. We estimate that the current collection covers ~75% of nonessential genes. We have used a portion of this collection to identify new genes required for the production of known virulence factors. Importantly, we have made strains available without restriction to the community via the Fungal Genetic Stock Center. In this amended renewal application, we will complete the Cryptococcus gene deletion collection, perform an additional round of quality- control checks including Southern hybridization, complete our screens for known virulence attributes, obtain reference profiles for fitness during infection, and then leverage our expertise, reagents and infrastructure to construct a strain collection in which each protein-coding gene is dual epitope-tagged, thereby enabling systematic studies of protein function. Completion of this work will have an enormous impact on the field by providing the research community with genomic resources not yet available for any human pathogen.

有效诊断和治疗危及人类生命的真菌感染是临床上一个重大未满足的问题主要人类真菌病原体的注释良好的基因组序列已可用于挑战。多年来，通过系统的应用创造了彻底改变医学真菌学的机会然而，为了发挥最大作用，全基因组序列需要与全基因组序列相结合。全基因组生物资源的可用性和标记基因集合。在剖析模型酵母酿酒酵母的基础生物学方面发挥了重要作用。前提是病原真菌中类似集合的产生将创造强大的实验能够采用综合方法来阐明哺乳动物宿主真菌毒力基础的工具并通过应用现有的工具来开发药物。体内和体外测定可对任何感兴趣过程中所有基因和蛋白质的行为进行功能分析。新型隐球菌是一种封装的芽殖酵母，是真菌性脑膜炎的最常见原因。据估计，每年约 1,000,000 例病例导致约 600,000 人死亡，占艾滋病患者死亡人数的三分之一。全世界范围内都有隐球菌性脑膜脑炎，这种重要的真菌病原体具有单倍体。遗传学和同源重组，使其成为优秀的模式病原体和模式生物。是 6967 个预测编码序列。在这笔资助的初始阶段，我们开发了一个信息学。基础设施、设计和购买了约 70,000 个寡核苷酸、招募/培训了人员并成功我们估计使用生物射弹（“基因枪”）转化构建并保藏了 4042 个基因敲除菌株。当前的集合涵盖了约 75% 的非必需基因，我们已使用该集合的一部分来识别产生已知毒力因子所需的新基因重要的是，我们已经制造了菌株。在本次修订后的更新中，社区可以不受限制地通过真菌遗传库存中心获得。申请后，我们将完成隐球菌基因缺失采集，进行额外一轮质量- 控制检查，包括 Southern 杂交，完成我们对已知毒力属性的筛选，获得感染期间的健康参考资料，然后利用我们的专业知识、试剂和基础设施构建一个菌株集合，其中每个蛋白质编码基因都带有双表位标记，从而使蛋白质功能的系统研究的完成将对该领域产生巨大的影响。为研究界提供尚无法用于任何人类病原体的基因组资源。