Development of a TILLING Resource for the Xenopus Research Community

描述(由申请人提供)： 在过去的几年里，几个模式生物小组使用测序方法，通过大量诱变动物(通常称为TILLING)进行筛选，以确定人们高度感兴趣的已知基因的突变。我们建议在伍兹霍尔的海洋生物实验室(MBL)开始为非洲爪哇研究社区开发耕作资源，选择这个地点有两个关键原因：1)即将在那里建造一个国家非洲爪哇资源中心(XRC)(用于饲养动物品系和高级研究培训)；以及2)在MBL建立一个与备受尊敬的约瑟芬湾保罗比较分子生物学和进化中心相关的先进测序设施。格兰杰实验室已经参与了为英国桑格中心的一个小规模耕作项目准备动物样本，在那里，使用常规测序，已经确定了ENU突变的非常近似的命中率。最近在这个筛选中发现了第一个零突变，在Rax基因(参与眼睛形成的关键调控基因)中，说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突变的更准确的剂量-反应曲线。其次，我们将收集数千只动物，为耕作分析提供材料。第三，我们将改进下一代通过耕作识别突变的方法，目的是识别几个关键调控基因的零突变。除了格兰杰实验室产生的突变动物外，霍卡和康隆实验室也将为这一努力提供样本。该项目将为开发一个更大的长期耕作项目提供基础，该项目将成为XRC的一部分，为全国范围内的研究人员提供产生突变系的资源。 公共卫生相关性： 项目简介这个项目的目标是确定对非洲爪蛙早期发育至关重要的基因突变。正在研究的基因在人类身上发生突变时，往往会导致基于基因的疾病，而对青蛙中这些突变的研究将为这些疾病提供新的见解，因为研究人员可以使用独特的工具来澄清这些动物的基因功能。

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.