High Throughput Cloning of Mutant C. elegans Loci

突变体秀丽隐杆线虫位点的高通量克隆

基本信息

批准号：
8432802
负责人：
BRUCE A BOWERMAN
金额：
$ 18.13万
依托单位：
UNIVERSITY OF OREGON
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：
2012-02-24 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8432802
关键词：
Alleles Animal Model Biological Process Caenorhabditis elegans Categories Cell Cycle Chemicals Classification Cloning Collection DNA DNA Sequence DNA lesion Development Embryo Embryonic Lethal Mutation Essential Genes Funding Genes Genetic Genetic Polymorphism Genetic Research Goals Gonadal structure Heating Human Investigation Laboratories Laboratory Research Lesion Malignant Neoplasms Maps Methods Morphogenesis Mutagenesis Mutation Nature Nematoda Organism Phenotype Population Predisposition Procedures RNA Interference Relative (related person)Research Research Personnel Resolution Single Nucleotide Polymorphism Map Site Staging Technology Temperature Time Tissues base cost effective egg gene function genome sequencing genome-wide human disease in vivo knock-down mutant next generation positional cloning therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): Conditional (heat-sensitive) mutations remain the most valuable genetic tool available for the in vivo investigation of essential gene requirements, and C. elegans is unique as an animal model in which one can feasibly isolate large numbers of rare conditional mutations. This proposal seeks to expand our identification of conditional mutations in essential C. elegans genes by (i) exploring previously ignored mutant phenotypes, and (ii) developing next generation DNA sequencing-based approaches to greatly reduce the time and labor required to map and positionally clone mutant C. elegans loci isolated after chemical mutagenesis screens. Specific Aim 1 focuses on the identification of 2000 new temperature-sensitive, embryonic-lethal C. elegans mutants; the systematic classification of all mutants into several phenotypic categories; the distribution of multiple mutant classes to collaborators; and the isolation of a large collection of mutants with previously unexplored gonad morphogenesis-defective phenotypes. Specific Aim 2 focuses on developing an Illumina DNA sequencing-based genome-wide approach to Single Nucleotide Polymorphism (SNP) mapping called Restriction-site Associated DNA polymorphism (RAD) mapping. To our knowledge, we are the first laboratory to explore applying this recently developed technology to the mapping of mutant loci in C. elegans. It promises to provide a high throughput and cost-effective approach to the mapping and positional cloning of large numbers of mutant loci. Specific Aim 3 explores Illumina DNA sequencing-based whole genome sequencing to identify the mutational lesions responsible for conditional embryonic-lethality in the mutants isolated in Aim 1 and mapped to small intervals in Aim 2. Our ultimate goal is to greatly expand the use of chemical mutagenesis screens to identify conditional mutations in essential C. elegans genes. By developing high throughput approaches to the positional cloning of conditionally mutant loci, our proposed exploratory research will substantially impact research by laboratories throughout the world that use C. elegans as an animal model for investigating many different and fundamentally important biological processes.

描述（由申请人提供）：条件（热敏感）突变仍然是可用于体内研究基本基因需求的最有价值的遗传工具，而秀丽隐杆线虫作为一种独特的动物模型，可以在其中分离出大量的罕见的条件突变。该提案旨在通过 (i) 探索以前被忽视的突变表型，以及 (ii) 开发下一代基于 DNA 测序的方法，以大大减少绘制和定位所需的时间和劳动力，从而扩大我们对秀丽隐杆线虫必需基因条件突变的识别。化学诱变筛选后分离出的克隆突变体秀丽隐杆线虫基因座。具体目标 1 重点鉴定 2000 个新的温度敏感、胚胎致死性秀丽隐杆线虫突变体；将所有突变体系统分类为几个表型类别；将多个突变类别分配给合作者；以及分离大量具有先前未探索的性腺形态发生缺陷表型的突变体。具体目标 2 重点开发一种基于 Illumina DNA 测序的全基因组方法来进行单核苷酸多态性 (SNP) 作图，称为限制性位点相关 DNA 多态性 (RAD) 作图。据我们所知，我们是第一个探索将这一最新开发的技术应用于线虫突变基因座定位的实验室。它有望为大量突变基因座的定位和定位克隆提供高通量和经济有效的方法。具体目标 3 探索基于 Illumina DNA 测序的全基因组测序，以识别在目标 1 中分离的突变体中导致条件性胚胎致死的突变损伤，并在目标 2 中映射到小区间。我们的最终目标是大大扩展化学物质的使用诱变筛选以识别秀丽隐杆线虫必需基因的条件突变。通过开发高通量方法来定位克隆条件突变位点，我们提出的探索性研究将极大地影响世界各地实验室的研究，这些实验室使用秀丽隐杆线虫作为动物模型来研究许多不同的、根本上重要的生物过程。