High Throughput Cloning of Mutant C. elegans Loci

描述(申请人提供)：条件性(热敏)突变仍然是体内研究基本基因需求的最有价值的遗传工具，线虫作为一种动物模型是独一无二的，在这种模型中，人们可以可行地分离大量罕见的条件性突变。这一建议旨在通过(I)探索以前被忽视的突变表型，以及(Ii)开发基于下一代DNA测序的方法来扩大我们对线虫基本基因条件突变的鉴定，以极大地减少定位和定位克隆化学诱变筛选后分离的线虫突变基因所需的时间和人力。具体目标1侧重于鉴定2000个新的温度敏感、胚胎致死的线虫突变体；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发现的性腺形态发生缺陷表型的突变体。具体目标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(SNP)作图方法，称为限制位点相关DNA多态(RAD)作图。据我们所知，我们是第一个探索将这项最近发展的技术应用于线虫突变基因定位的实验室。它有望为大量突变基因座的定位和定位克隆提供一种高通量和低成本的方法。特异性目标3探索了基于Illumina DNA测序的全基因组测序，以确定在Aim 1中分离的突变株中导致条件性胚胎致死的突变损伤，并在Aim 2中定位到较小的间隔。我们的最终目标是极大地扩大化学诱变筛选的使用，以鉴定线虫基本基因的条件突变。通过开发高通量的位置克隆条件突变基因座的方法，我们拟议的探索性研究将对世界各地使用线虫作为动物模型来研究许多不同的和基本重要的生物过程的实验室的研究产生重大影响。 公共卫生相关性：线虫秀丽线虫作为一种动物模型是独一无二的，在这种动物模型中，人们可以可行地分离基本基因中大量罕见的条件性突变。条件性(热敏)突变仍然是研究基本基因需求的最有价值的遗传工具，这些基因需求往往具有多个基本需求，在许多情况下，甚至在单个细胞分裂周期中也是如此。此外，秀丽线虫的基本基因在包括人类在内的其他生物中被广泛保守，而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和能力直接相关。通过促进大量这些用于研究保守和必要基因的宝贵遗传工具的分离，我们将大大有助于我们对世界各地实验室正在研究的许多基本生物学过程的基本理解。研究秀丽线虫等模式生物的生物学过程对于了解人类疾病机制以及确定可能的治疗靶点和机会具有重要意义。

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.