TILLING the Zebrafish Genome: A Reverse Genetic Approach

描述（由申请人提供）：斑马鱼已成为越来越多的研究人员选择的模型系统，这些研究人员对了解脊椎动物发育、疾病和进化机制感兴趣。这项多主要研究者提案的三位主要研究者各自建立了靶向基因组局部病变（TILLING）方法，用于使用Cell 1检测来鉴定斑马鱼特定感兴趣基因中的N-乙基-N-亚硝基脲（ENU）诱导突变，并使用这种方法来鉴定无义或剪接位点突变，预测43个基因的功能强烈或完全丧失。该方法包括在我们每个实验室独立建立的随机ENU诱变的冷冻保存鱼的大型文库中筛选独特的突变。我们希望将这些宝贵的资源提供给斑马鱼社区。在这项为期3年的资助中，我们建议在斑马鱼社区成员感兴趣的120个基因中鉴定功能丧失突变（定义为产生过早终止密码子或破坏剪接位点的突变），并通过提交给斑马鱼国际资源中心（ZIRC）提供。在三个群体之间建立TILLING联合体减少了冗余的工作，并增加了识别靶基因中有害突变的机会。然而，单独地，我们的文库被预测在我们筛选的基因中含有20%至58%的功能丧失突变，组合起来，它们被预期在超过80%的靶中含有功能丧失突变。在目标1中，我们建议在三个位置依次筛选靶基因，直到鉴定出一个或多个功能丧失突变。潜在的TILLING目标将由社区成员提交，并由一个由10名成员组成的外部咨询委员会根据高生物医学相关性和其他反向遗传学方法的不可访问性等标准进行排名。在目标2中，我们建议恢复这些突变体，进行初步的表型表征，并首先将其提供给请求者，然后在恢复后六个月内，通过ZIRC向更广泛的斑马鱼社区提供。最后，在目标3中，我们建议探索从我们的ENU诱变文库中PCR扩增靶点的大规模并行测序，作为我们目前TILLING方法的更高通量替代方案。 公共卫生相关性：脊椎动物模式生物，如斑马鱼，其中基因功能可以通过详细分析突变表型来理解，为人类发育和疾病的机制提供了重要的见解。在过去的资助期间，我们采用了TILLING，一种在斑马鱼中发现感兴趣基因突变的方法，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找到功能丧失突变。我们现在建议使用TILLING来识别对生物医学研究具有重要意义的120个基因中的突变，并尽快将这些突变体提供给斑马鱼社区。

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.