Development of a TILLING Resource for the Xenopus Research Community

为非洲爪蟾研究界开发 TILLING 资源

• 批准号: 8131817
• 负责人: Robert M Grainger
• 金额: $ 19.07万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-20 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8131817
• 关键词: Animal Housing; Animal Model; Animals; Back; Biological; Biological Models; Cell physiology; Collaborations; Communities; Complement; DNA; DNA Sequence; DNA Sequence Analysis; Development; Developmental Process; Disease; Dose; Ethylnitrosourea; Eye Development; Funding; Future; Genes; Genome; Genomics; Goals; Grant; Homeobox Genes; Human; Induced Mutation; Informatics; Label; Laboratories; Lesion; Marines; Methods; Molecular Biology; Molecular Evolution; Mutagenesis; Mutagens; Mutate; Mutation; Phase; Phenotype; Population; Preparation; Protocols documentation; Rana; Regulator Genes; Research; Research Personnel; Research Training; Resources; Sampling; Scheme; Sequence Analysis; Site; Spermatogonia; Technology; United States National Institutes of Health; Wood material; Work; Xenopus; animal resource; base; comparative; ethylurea; eye formation; gene function; high reward; high risk; insight; interest; male; mutant; next generation; novel; null mutation; offspring; public health relevance; research study; response; sound; sperm cell; tool; xenopus genome

DESCRIPTION (provided by applicant): During the past several years several model organism groups have used sequencing methods to screen through large populations of mutagenized animals (commonly referred to as TILLING) to identify mutations in known genes of high interest. We propose here to initiate development of a TILLING resource for the Xenopus research community at the Marine Biological Laboratory (MBL) at Woods Hole, a site chosen for two key reasons: 1) the impending construction of a national Xenopus Resource Center (XRC) there (for housing animals lines and advanced research training); and 2) the presence of an advanced sequencing facility associated with the highly regarded Josephine Bay Paul Center for Comparative Molecular Biology and Evolution at the MBL. The Grainger lab has already been involved in preparing animal samples for a small scale TILLING project at the Sanger Centre in the UK, where, using conventional sequencing, a very approximate hit rate for ENU mutagenesis has been determined. Recently the first null mutation has been identified in this screen, in the Rax gene (a key regulatory gene involved in eye formation), illustrating that the basic strategy is effective. In this proposal we have three goals. First we will use next generation sequencing to ascertain a far more accurate dose- response curve for ENU mutagenesis. Second, we will collect several thousand animals to provide material for a TILLING analysis. Third, we will refine next generation approaches for identifying mutations by TILLING, with the aim of identifying null mutations in several key regulatory genes. In addition to mutant animals generated in the Grainger lab, the Khokha and Conlon labs will also contribute samples to this effort. This project will provide the basis for development of a larger, long term TILLING project that will become part of the XRC, providing a resource for generating mutant lines for investigators nationwide. PUBLIC HEALTH RELEVANCE: Project Narrative The goal of this project is to identify mutations in genes important for the early development of the frog Xenopus. The genes under study, when mutated in humans, often result in genetically based diseases, and study of these mutations in the frog will provide new insights about these diseases because of the unique tools available to researchers for clarifying gene function in these animals.

描述（由申请人提供）： 在过去的几年中，几个模型生物组已使用测序方法对大量诱变动物进行筛选（通常称为 TILLING），以识别高度感兴趣的已知基因中的突变。我们在此建议在伍兹霍尔的海洋生物实验室 (MBL) 启动为非洲爪蟾研究界开发 TILLING 资源，选择该地点有两个主要原因：1) 即将在那里建设国家非洲爪蟾资源中心 (XRC)（用于饲养动物品系和高级研究培训）； 2) MBL 拥有与备受推崇的 Josephine Bay Paul 比较分子生物学和进化中心相关的先进测序设施。 Grainger 实验室已经参与了英国桑格中心小规模 TILLING 项目的动物样品准备工作，该项目使用传统测序，确定了 ENU 诱变的非常近似的命中率。最近，在该筛选中，在 Rax 基因（参与眼睛形成的关键调控基因）中发现了第一个无效突变，这表明基本策略是有效的。在这个提案中，我们有三个目标。首先，我们将使用下一代测序来确定 ENU 诱变的更准确的剂量反应曲线。其次，我们将收集数千只动物，为 TILLING 分析提供材料。第三，我们将完善下一代通过 TILLING 识别突变的方法，目的是识别几个关键调控基因的无效突变。除了 Grainger 实验室产生的突变动物外，Khokha 和 Conlon 实验室也将为这项工作提供样本。该项目将为开发更大的长期 TILLING 项目奠定基础，该项目将成为 XRC 的一部分，为全国研究人员生成突变株系提供资源。 公共卫生相关性： 项目叙述该项目的目标是识别对青蛙爪蟾早期发育重要的基因突变。所研究的基因在人类中发生突变时，通常会导致基于遗传的疾病，而对青蛙中这些突变的研究将为研究人员提供关于这些疾病的新见解，因为研究人员可以使用独特的工具来阐明这些动物的基因功能。