TILLING the Zebrafish Genome: A Reverse Genetic Approach

描述(申请人提供)：斑马鱼已经成为越来越多对了解脊椎动物发育、疾病和进化机制感兴趣的研究人员选择的模型系统。这项由多名首席调查员组成的提案的三位主要研究人员各自建立了靶向基因组局部损伤(TILLING)方法，用于利用CEL1检测来鉴定N-乙基-N-亚硝脲(ENU)诱导的斑马鱼特定基因的突变，并使用这种方法来识别预测43个基因强烈或完全丧失功能的无义或剪接位点突变。该方法包括在我们每个实验室独立建立的随机ENU诱变的冷冻保存鱼类的大型文库中筛选独特的突变。我们希望向斑马鱼界提供这些宝贵的资源。在这项为期3年的拨款中，我们建议识别斑马鱼群落成员感兴趣的120个基因中的功能丧失突变(为我们的目的定义为产生过早终止密码子或破坏剪接点的突变)，并通过提交给斑马鱼国际资源中心(ZIRC)提供这些突变。在这三个小组之间建立一个耕作联盟可以减少重复的工作，并增加识别目标基因有害突变的机会。另外，我们的文库预计在我们筛选的基因中包含20%到58%的功能丧失突变，它们预计在超过80%的靶基因中包含功能丧失突变。在目标1中，我们建议在三个位置顺序筛选靶基因，直到发现一个或多个功能丧失突变。潜在的耕作目标将由社区成员提交，并将由一个由10名成员组成的外部顾问委员会根据高度生物医学相关性和无法获得其他反向遗传学方法等标准进行排名。在目标2中，我们建议恢复这些突变体，进行初步的表型特征，并首先将它们提供给请求者，然后在恢复后的六个月内，通过Zirc提供给更广泛的斑马鱼群落。最后，在目标3中，我们建议探索从我们的ENU诱变文库中大规模并行测序PCR扩增的目标，作为我们目前的耕作方法的一种更高吞吐量的替代方案。 公共卫生相关性：脊椎动物模式生物，如斑马鱼，可以通过对突变表型的详细分析来了解基因功能，为人类发育和疾病的机制提供了重要的见解。在过去的拨款期间，我们对TILLING进行了改造，这是一种寻找感兴趣基因突变的方法，适用于斑马鱼，并建立了资源，使我们能够在斑马鱼基因组中的大部分基因中发现功能丧失的突变。我们现在建议使用TILLING来识别对生物医学研究重要的120个基因的突变，并尽快将这些突变提供给斑马鱼群体。

DESCRIPTION (provided by applicant): The zebrafish has become the model system of choice for a growing number of investigators interested in understanding mechanisms of vertebrate development, disease and evolution. The three principal investigators on this multiple-Principal Investigator proposal have each established the Targeting Local Lesions IN Genomes (TILLING) methodology for identifying N-ethyl-N-nitrosourea (ENU)-induced mutations in specific genes of interest in zebrafish using Cel1 detection, and have used this approach to identify nonsense or splice site mutations predictive of strong or complete loss of function of 43 genes. The approach involves screening for unique mutations in a large library of randomly ENU-mutagenized, cryopreserved fish that has been built independently in each of our labs. We wish to make these valuable resources available to the zebrafish community. In this 3-year grant, we propose to identify loss-of-function mutations (defined for our purposes as mutations that create premature stop codons or that disrupt splice sites) in 120 genes of interest to the members of the zebrafish community and to make them available via submission to the Zebrafish International Resource Center (ZIRC). Establishing a TILLING consortium between the three groups reduces redundancy of effort and increases the chances of identifying deleterious mutations in target genes. Whereas separately, our libraries are predicted to contain loss-of-function mutations in between 20% and 58% of the genes we screen, combined, they are expected to contain loss-of-function mutations in over 80% of targets. In Aim 1 we propose to screen target genes sequentially at the three locations until one or more loss-of-function mutations are identified. Potential TILLING targets will be submitted by members of the community and will be ranked by a 10-member external advisory board according to criteria such as high biomedical relevance and inaccessibility to other reverse genetics methods. In Aim 2 we propose to recover these mutants, to do a preliminary phenotypic characterization and to provide them first to the requester and then, within six months of recovery, to the wider zebrafish community via ZIRC. Finally, in Aim 3 we propose to explore massively parallel sequencing of PCR-amplified targets from our ENU-mutagenized libraries as a higher-throughput alternative to our current TILLING methodology. PUBLIC HEALTH RELEVANCE: Vertebrate model organisms such as the zebrafish, in which gene function can be understood through the detailed analysis of mutant phenotypes, provide important insights into mechanisms of human development and disease. In the past grant period we adapted TILLING, a methodology to find mutations in genes of interest, to the zebrafish, and built resources that can allow us to find loss-of-function mutations in a large fraction of the genes in the zebrafish genome. We now propose to use TILLING to identify mutations in 120 genes that are of importance to biomedical research, and to make these mutants available to the zebrafish community as rapidly as possible.