International Zebrafish Mutagenic Protein Trap

国际斑马鱼诱变蛋白陷阱

• 批准号: 8145515
• 负责人: Stephen Carl Ekker
• 金额: $ 56.97万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8145515
• 关键词: Alleles; Animal Model; Archives; Area; Behavior; Behavioral; Behavioral Genetics; Biochemical Pathway; Biological; Biological Models; Biological Process; Biology; Cataloging; Catalogs; Chromosome Mapping; Clinic; Collection; Communities; Complex; Computer Simulation; Core Facility; Cryopreservation; Data; Databases; Development; Disease; Equipment and supply inventories; Evolution; Fishes; Fostering; Gene Expression Profile; Genes; Genetic; Genome; Genomics; Homologous Gene; Human; International; Laboratories; Messenger RNA; Microtus; Modeling; Molecular; Mus; Mutagens; Mutation; Nematoda; Neural Crest; Nicotine; Nuclear; Organogenesis; Pattern; Process; Proteins; Proteome; Protocols documentation; Research; Research Personnel; Resources; Role; Shipping; Ships; Signal Pathway; System; Techniques; Time; United States National Institutes of Health; Vascular Endothelial Growth Factors; Vertebrate Biology; Vertebrates; Zebrafish; Zebrafish Proteins; base; fly; genetic linkage analysis; genome database; human disease; in vivo; insight; loss of function; mutant; next generation; phenome; response; sperm cell; symposium; tool; vertebrate genome; zebrafish genome

DESCRIPTION (provided by applicant): The zebrafish (Danio rerio) is the preeminent non-mammalian vertebrate system for the study of core vertebrate biology and behavior and for the modeling of human disease. Collections of molecularly characterized mutant lines have been powerful enabling tools for the nematode, fly and mouse fields. Genomic approaches have robustly characterized the nuclear genome and transcriptome. However, the genomic assessment of the full complexity of the proteome in a dynamic context and in vivo is still largely unknown. The zebrafish offers the first comprehensive analysis of the proteome using as template an entire vertebrate genome. We show that mutagenic protein trap gene-breaking transposons (GBTs) are effective and conditional loss- of-function tools for exploring traditional areas such as the biology of development (including organogenesis) but also open up new options including the genetics of behavior such as the biology of the nicotine response. This application is in response to "PAR-08-139 Enhancing Zebrafish Research with Research Tools and Techniques (R01)" to establish a large-scale collection of these conditional mutant alleles for the zebrafish community through a coordinated international effort. We will deliver the following at project's end: I. 2000 new mutagenic protein trap GBT zebrafish lines with basic description of the tagged expression patterns will be established and made available in real-time through the searchable online database zfishbook.org. These lines are immediately available through distribution by the Mayo Clinic Zebrafish Core Facility. II. We will conduct molecular analyses of these lines and subsequent annotation on the zebrafish genome project to identify the trapped proteins in this collection. We will initially expand the use of established 5' RACE protocols to identify trapped protein-encoding mRNA. We will subsequently engage next generation sequence techniques followed by linkage analysis to potentially accelerate the process of mapping GBT insertions. All sequence information will be initially distributed through zfishbook.org and then integrated in ZFIN, Fishmap and other zebrafish genomic databases. III. We will regularly synchronize this collection with the Zebrafish International Resource Center (ZIRC) for long-term archival and distribution to the zebrafish community. We will use a recently established 2D barcoding inventory system with a state-of-the-art sperm cryopreservation approach to facilitate regular shipping of new lines to ZIRC. This proposal is to establish a molecularly characterized collection, in silico catalog and distribution network of conditional mutations for the preeminent non-mammalian vertebrate, the zebrafish. These dominantly marked mutant lines also represent an ideal substrate for large-scale behavioral and other phenotypic assessment of the genome as a part of the recently launched phenome project for the zebrafish. PUBLIC HEALTH RELEVANCE: This proposal is to generate a mutant resource of 2000 new conditional protein trap mutations for the zebrafish (Danio rerio), the preeminent non-mammalian system for the study of core vertebrate biology and for modeling of human disease. These fish lines are available in real-time through distribution by the Mayo Clinic Zebrafish Core Facility. The full collection will be established through a coordinated international effort and archived and distributed by the Zebrafish International Resource Center for use by all zebrafish researchers worldwide.

描述（由申请人提供）：斑马鱼（Danio rerio）是研究核心脊椎动物生物学和行为以及人类疾病建模的卓越的非哺乳动物脊椎动物系统。分子特征突变系的收集已经成为线虫、苍蝇和老鼠领域的有力工具。基因组学方法已经强有力地表征了核基因组和转录组。然而，在动态环境和体内对蛋白质组完整复杂性的基因组评估仍然很大程度上是未知的。斑马鱼提供了第一个以整个脊椎动物基因组为模板的蛋白质组综合分析。我们发现诱变蛋白诱捕基因断裂转座子（gbt）是探索传统领域（如发育生物学（包括器官发生））的有效和有条件的功能丧失工具，但也开辟了新的选择，包括行为遗传学，如尼古丁反应生物学。该申请是为了响应“PAR-08-139使用研究工具和技术加强斑马鱼研究（R01）”，通过协调的国际努力，为斑马鱼群落建立这些条件突变等位基因的大规模收集。在项目结束时，我们将提供以下内容：1 .建立2000个新的诱变蛋白捕获GBT斑马鱼系，并对标记的表达模式进行基本描述，并通过可搜索的在线数据库zfishbook.org实时提供。通过梅奥诊所斑马鱼核心设施的分发，这些线路立即可用。2。我们将对这些细胞系进行分子分析，并对斑马鱼基因组计划进行后续注释，以鉴定该集合中的捕获蛋白。我们将首先扩大已建立的5' RACE协议的使用，以鉴定捕获的蛋白质编码mRNA。随后，我们将采用下一代序列技术，随后进行连锁分析，以潜在地加速定位GBT插入的过程。所有序列信息将首先通过zfishbook.org发布，然后整合到ZFIN、Fishmap等斑马鱼基因组数据库中。3。我们将定期与斑马鱼国际资源中心（ZIRC）同步收集这些资料，以便长期存档并分发给斑马鱼群落。我们将使用最近建立的二维条形码库存系统，采用最先进的精子冷冻保存方法，以方便定期向ZIRC运送新线路。这一建议是建立一个分子特征的收集，在硅目录和条件突变的分布网络为卓越的非哺乳动物脊椎动物，斑马鱼。作为最近启动的斑马鱼表型计划的一部分，这些显性标记突变系也代表了大规模行为和其他基因组表型评估的理想底物。