TILLING the Zebrafish Genome: A Reverse Genetic Approach

描述（由申请人提供）：斑马鱼已经成为越来越多对理解脊椎动物发育、疾病和进化机制感兴趣的研究人员选择的模型系统。在这个多负责人提案中，三位主要研究者分别建立了靶向局部病变基因组（TILLING）方法，用于使用Cel1检测鉴定斑马鱼中n -乙基-n -亚硝基脲（ENU）诱导的特定基因突变，并使用这种方法鉴定无义或剪接位点突变，预测43个基因的强烈或完全功能丧失。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，我们建议在斑马鱼群落成员感兴趣的120个基因中确定功能丧失突变（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）提供这些突变。在三个小组之间建立TILLING联盟减少了工作的冗余，并增加了识别靶基因中有害突变的机会。然而，我们的文库预计在我们筛选的基因中包含20%到58%的功能缺失突变，而综合起来，它们预计在超过80%的目标中包含功能缺失突变。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10人组成的外部咨询委员会根据诸如生物医学相关性高和其他反向遗传学方法不可及性等标准进行排名。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Aim 3中，我们建议从我们的enu突变文库中探索pcr扩增目标的大规模平行测序，作为我们当前TILLING方法的更高通量替代方案。

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.