Zebrafish Mutant Mapping Facility

描述(申请人提供)：这项建议的目的是加速鉴定在诱变筛选过程中发现的斑马鱼基因。这项建议是对PAR-02-142关于开发“斑马鱼遗传研究工具”的响应。这种PA的一个主要焦点是使用新的筛选来识别感兴趣的突变体。然而，这项工作的一个重要部分是确定导致产生新表型的基因。虽然已经描述了数千个突变，但斑马鱼数据库(ZFIN)中只列出了167个已知突变的基因(尽管可能还有一些没有列出)。其主要原因是必须使用位置克隆策略的耗时特性。通过位置克隆发现基因的主要瓶颈是需要进行全基因组连锁图谱。这需要相当多的资源和时间，以及技术专长。这项提议的目标是通过建立一个高通量的测绘设施来消除这一瓶颈。通常繁重的任务--筛选数百个标记以找到与感兴趣的突变相关的标记--将在两天内完成。这将使用荧光测序仪自动对跨越基因组的SSLP(短序列长度多态)进行基因分型。映射将分两个主要阶段进行。首先，将使用整体偏析分析确定大致位置。其次，一旦确定了紧邻的标记，将对500-1000个突变胚胎进行基因分型，以确定包含突变基因的区域。第三，对于少数突变体，基因将被识别出来。CORE每年将绘制至少100个突变的图谱，并在5-10个月内识别该基因，尽管一旦基因组序列完成，这两个数字都可能大幅增加。这项服务将是模块化的，这样研究人员就可以由该设施完成初始绘制阶段，然后在自己的实验室完成突变基因分离。该项目将产生相当大的影响，因为它将极大地加速发现已经被证明是人类疾病模型的基因。该设施致力于完全开放使用，所有与测绘有关的数据都将在出版时提供。鉴于斑马鱼作为模式生物的重要性，这一设施将立即对发现突变基因的速度产生影响，这将为研究与疾病有关的重要生化途径的遗传基础提供新的见解。

DESCRIPTION (provided by applicant): The objective of this proposal is to accelerate the identification of zebrafish genes discovered during mutagenesis screens. This proposal is in response to PAR-02-142 to develop "Tools for Genetic Studies in Zebrafish." A major focus of this PA is to use novel screens to identify mutants of interest. However, an essential part of this effort is to identify the genes responsible for the novel phenotypes generated. While thousands of mutants already have been described, only 167 genes are listed in the zebrafish database (ZFIN) for which mutations are known (although there are probably some not listed). The major reason for this is the time consuming nature of the positional cloning strategy that must be used. The major bottleneck in gene discovery by positional cloning is the need to do whole genome linkage mapping. This takes considerable resources and time, as well as technical expertise. This goal of this proposal is to eliminate this bottleneck by setting up a high throughput mapping facility. The usual laborious task of screening sometimes hundreds of markers to find one that is linked to the mutation of interest will be accomplished in 2 days. This will be done using fluorescence sequencers to automate genotyping of SSLPs (short sequence length polymorphisms) that span the genome. The mapping will be done in two main stages. First, an approximate location will be determined using bulk segregant analyses. Second, once closely flanking markers are identified 500-1000 individual mutant embryos will be genotyped to identify the region containing the mutant gene. Third, for a small number of mutants the genes will be identified. The core will map a minimum of 100 mutants per year and identify the gene in 5-10, although both these numbers are likely to increase dramatically once the genome sequence is completed. The service will be modular so that investigators can have the initial mapping phases done by the facility, and then complete the mutant gene isolation in there own laboratories. This project will have considerable impact because it will greatly accelerate the discovery of genes that already have been shown to be models of human diseases. The facility is committed to full and open access and all data pertaining to mapping will be made available at publication. Given the importance of zebrafish as a model organism this facility will have an immediate impact on the rate at which mutant genes are discovered, which will provide new insights into the genetic underpinnings of important biochemical pathways involved in disease.