Development of a TILLING Resource for the Xenopus Research Community

描述（由申请人提供）： 在过去的几年中，几个模式生物群体已经使用测序方法来筛选大量的诱变动物（通常称为TILLING），以鉴定高度关注的已知基因中的突变。我们建议在伍兹霍尔的海洋生物实验室（MBL）为非洲爪蟾研究社区开发TILLING资源，选择该地点有两个关键原因：1）即将在那里建设国家非洲爪蟾资源中心（XRC（用于圈养动物和高级研究培训）;以及2）与MBL的约瑟芬湾保罗比较分子生物学和进化中心（Josephine Bay Paul Center for Comparative Molecular Biology and Evolution）相关的先进测序设施的存在。Grainger实验室已经参与了英国桑格中心小规模TILLING项目的动物样本制备，在该项目中，使用常规测序，已经确定了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）中鉴定了第一个无效突变，说明基本策略是有效的。在这项建议中，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，我们将收集几千只动物，为TILLING分析提供材料。第三，我们将完善下一代TILLING识别突变的方法，目的是识别几个关键调控基因中的无效突变。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。该项目将为开发一个更大的长期TILLING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株系的资源。 公共卫生相关性： 这个项目的目标是识别对蛙爪蟾早期发育重要的基因突变。研究中的基因在人类中发生突变时，通常会导致遗传性疾病，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澄清这些动物中的基因功能。

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.