Zebrafish Mutant Mapping Facility

斑马鱼突变体绘图设施

• 批准号: 7119641
• 负责人: RONALD G GREGG
• 金额: $ 21.53万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7119641
• 关键词: alternatives to animals in research; animal colony; animal population genetics; fluorescent dye /probe; gene expression; genetic mapping; genetic markers; genetic recombination; genetic screening; genotype; high throughput technology; linkage mapping; molecular cloning; mutant; polymerase chain reaction; zebrafish

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.

描述（由申请人提供）：本提案的目的是加速在诱变筛选过程中发现的斑马鱼基因的鉴定。本提案是对PAR-02-142的回应，旨在开发“斑马鱼遗传研究工具”。“该PA的一个主要重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负责产生新表型的基因。虽然已经描述了数千种突变体，但在斑马鱼数据库（ZFIN）中仅列出了167个已知突变的基因（尽管可能有一些未列出）。其主要原因是必须使用的位置克隆策略的耗时性质。通过定位克隆发现基因的主要瓶颈是需要进行全基因组连锁图谱。这需要大量的资源和时间以及技术专长。该提案的目标是通过建立高吞吐量映射设施来消除这一瓶颈。通常，筛选数百个标记以找到与感兴趣的突变相关的标记的繁重任务将在2天内完成。这将使用荧光测序仪来完成，以自动进行跨越基因组的SSLP（短序列长度多态性）的基因分型。测绘工作将分两个主要阶段进行。首先，将使用整体分离子分析确定大致位置。第二，一旦鉴定出紧密侧翼标记，将对500-1000个个体突变胚胎进行基因分型以鉴定含有突变基因的区域。第三，对于少数突变体，将鉴定基因。核心每年将绘制至少100个突变体，并在5-10个中识别基因，尽管一旦基因组序列完成，这两个数字可能会急剧增加。该服务将是模块化的，以便研究人员可以在设施中完成初始映射阶段，然后在自己的实验室中完成突变基因分离。该项目将产生相当大的影响，因为它将大大加速发现已经被证明是人类疾病模型的基因。该设施致力于充分和开放的访问，所有有关地图的数据将在出版时提供。鉴于斑马鱼作为模式生物的重要性，该设施将对突变基因的发现率产生直接影响，这将为疾病所涉及的重要生化途径的遗传基础提供新的见解。