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

• 批准号: 8224249
• 负责人: BRUCE A BOWERMAN
• 金额: $ 21.53万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 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/8224249
• 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

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. PUBLIC HEALTH RELEVANCE: The nematode Caenorhabditis elegans is unique as an animal model in which one can feasibly isolate large numbers of rare conditional mutations in essential genes. Conditional (heat-sensitive) mutations remain the most valuable genetic tool available for the investigation of essential gene requirements, which often have multiple essential requirements, in many cases even during a single cell division cycle. Moreover, essential C. elegans genes are with few exceptions widely conserved in other organisms including humans, and are in many cases of direct relevance to our understanding of and ability to detect and treat cancers and other important human diseases. By promoting the isolation of large collections of these valuable genetic tools for the study of conserved and essential genes, we will contribute substantially to our basic understanding of numerous fundamental biological processes under investigation in laboratories throughout the world. The investigation of biological processes in model organisms like C. elegans is of fundamental importance for understanding human disease mechanisms, and for identifying possible therapeutic targets and opportunities.

描述（申请人提供）：条件（热敏）突变仍然是体内研究必需基因需求的最有价值的遗传工具，C。秀丽线虫作为动物模型是独特的，其中人们可以可行地分离大量罕见的条件突变。该提案旨在扩大我们对必需C中条件突变的识别。通过（i）探索以前被忽视的突变表型，和（ii）开发下一代基于DNA测序的方法，以大大减少映射和定位克隆突变C所需的时间和劳动力，来研究线虫基因。化学诱变筛选后分离的elegans基因座。 具体目标1的重点是鉴定2000个新的温度敏感的，胚胎致死的C。线虫突变体;将所有突变体系统地分类为几个表型类别;将多个突变体类别分配给合作者;以及分离大量具有以前未探索的性腺形态发生缺陷表型的突变体。 Specific Aim 2专注于开发一种基于Illumina DNA测序的全基因组单核苷酸多态性（SNP）作图方法，称为限制性位点相关DNA多态性（RAD）作图。据我们所知，我们是第一个实验室，探索应用这一最近开发的技术，以定位突变位点的C。优雅的它有望提供一个高通量和成本效益的方法来定位和克隆大量的突变基因座。 特定目标3探索了基于Illumina DNA测序的全基因组测序，以鉴定在目标1中分离的突变体中导致条件性胚胎致死性的突变病变，并在目标2中映射到小间隔。 我们的最终目标是极大地扩展化学诱变筛选的使用，以确定必需C的条件突变。elegans基因通过开发条件突变基因座定位克隆的高通量方法，我们提出的探索性研究将对世界各地使用C.线虫作为研究许多不同的和根本重要的生物过程的动物模型。 公共卫生相关性：线虫秀丽隐杆线虫是一种独特的动物模型，其中可以可行地分离大量的罕见的条件突变的必需基因。条件（热敏）突变仍然是最有价值的遗传工具，可用于研究必需基因的要求，这往往有多个基本要求，在许多情况下，甚至在一个细胞分裂周期。此外，必要的C。除了少数例外，秀丽线虫的基因在包括人类在内的其他生物体中广泛保守，并且在许多情况下与我们对癌症和其他重要人类疾病的理解以及检测和治疗癌症和其他重要人类疾病的能力直接相关。通过促进这些宝贵的遗传工具的大量收集的保守和必需的基因的研究的隔离，我们将大大有助于我们的许多基本的生物过程的基本了解正在调查中的实验室在世界各地。在模式生物如C.线虫对于理解人类疾病机制以及确定可能的治疗靶点和机会具有根本重要性。