Cryptococcus knockout and tag resource

隐球菌敲除和标签资源

• 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
• 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

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生命的人类真菌感染是一个主要的未满足的临床 挑战.人类主要真菌病原体的注释良好的基因组序列已经可用于 多年来，创造了一个机会，通过系统的应用，彻底改变医学真菌学 全基因组方法。然而，为了最大限度地发挥作用，全基因组序列需要与 全基因组生物资源。全基因组敲除和标记基因集合的可用性 在解剖模式酵母酿酒酵母的基础生物学方面起了重要作用。我们 前提是，在病原真菌中产生类似的集合将产生强大的实验， 使综合方法能够阐明哺乳动物宿主中真菌毒力基础的工具 and to develop发展drugs药物.这些工具通过应用现有的 体内和体外测定以功能性地描述所有基因和蛋白质在任何感兴趣的过程中的行为。 新型隐球菌是一种被包裹的芽殖酵母，是真菌性脑膜炎最常见的原因。 据估计，每年约有100万例病例导致约60万人死亡。三分之一的艾滋病患者死亡 是隐球菌脑膜脑炎的结果。这种重要的真菌病原体具有单倍体 遗传学和同源重组，使其成为一个很好的模式病原体和模式生物。那里 是6967个预测编码序列。在这项资助的最初阶段，我们开发了一种信息学， 基础设施，设计和购买约70，000个寡核苷酸，招募/培训人员，并成功地 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。我们估计 目前的收集涵盖了约75%的非必需基因。我们用这些藏品的一部分 确定产生已知毒力因子所需的新基因。重要的是，我们已经制造出 可通过真菌遗传库存中心不受限制地向社区提供。在这次更新中， 申请后，我们将完成隐球菌基因缺失的采集，再进行一轮质量检测- 包括Southern杂交在内的控制检查，完成已知毒力属性的筛选，获得 在感染过程中参考健康状况，然后利用我们的专业知识，试剂和基础设施， 构建其中每个蛋白质编码基因被双表位标记的菌株集合，从而使得 蛋白质功能的系统研究。这项工作的完成将对实地产生巨大影响， 为研究界提供了尚未获得的任何人类病原体的基因组资源。