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.
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.
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