High Throughput Cloning of Mutant C. elegans Loci

描述（由申请人提供）：条件（热敏感）突变仍然是体内基本基因需求研究中最有价值的遗传工具，秀丽隐杆线虫是一种独特的动物模型，人们可以在其中分离出大量罕见的条件突变。本研究旨在通过(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克隆化学诱变筛选后分离的秀丽隐杆线虫突变位点所需的时间和劳动力，扩大我们对秀丽隐杆线虫必要基因条件突变的鉴定。特异性目标1侧重于鉴定2000种新的温度敏感、胚胎致死性秀丽隐杆线虫突变体；所有突变体的系统分类为几个表型类别；多突变类在合作者中的分布；并且分离了大量以前未被发现的性腺形态发生缺陷表型的突变体。具体目标2侧重于开发基于Illumina DNA测序的全基因组单核苷酸多态性（SNP）定位方法，称为限制性位点相关DNA多态性（RAD）定位。据我们所知，我们是第一个探索将这项最新开发的技术应用于线虫突变位点定位的实验室。它有望为大量突变位点的定位和克隆提供一种高通量和低成本的方法。特异性Aim 3探索基于Illumina DNA测序的全基因组测序，以确定在Aim 1中分离的突变体中负责条件胚胎致死的突变病变，并在Aim 2中定位到小间隔。我们的最终目标是大大扩大化学诱变筛选的使用，以确定秀丽隐杆线虫基本基因的条件突变。通过开发条件突变位点的高通量定位克隆方法，我们提出的探索性研究将极大地影响世界各地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和根本重要的生物过程的研究。

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.