International Zebrafish Mutagenic Protein Trap

描述（由申请人提供）：斑马鱼（Danio rerio）是用于研究核心脊椎动物生物学和行为以及用于人类疾病建模的杰出的非哺乳动物脊椎动物系统。分子特征的突变株系的集合已经成为线虫、蝇和小鼠领域的有力工具。基因组学方法已经有力地表征了核基因组和转录组。然而，在动态背景下和体内的蛋白质组的完整复杂性的基因组评估仍然是未知的。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。 我们表明，诱变蛋白陷阱基因破坏转座子（GBT）是有效的和条件性功能丧失的工具，用于探索传统领域，如发育生物学（包括器官发生），但也开辟了新的选择，包括行为遗传学，如尼古丁反应的生物学。本申请是对“PAR-08-139用研究工具和技术加强斑马鱼研究（R 01）”的响应，旨在通过协调的国际努力为斑马鱼社区建立这些条件突变等位基因的大规模收集。我们将在项目结束时提供以下内容：I.将建立2000个新的诱变蛋白陷阱GBT斑马鱼品系，并通过可搜索的在线数据库zfishbook.org实时提供标记表达模式的基本描述。这些品系通过马约诊所斑马鱼核心设施的分发立即可用。二.我们将对这些品系进行分子分析，并随后对斑马鱼基因组计划进行注释，以确定这些集合中的捕获蛋白。我们最初将扩大使用已建立的5' RACE方案来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，然后进行连锁分析，以潜在地加速绘制GBT插入的过程。所有序列信息最初将通过zfishbook.org发布，然后整合到ZFIN、Fishmap和其他斑马鱼基因组数据库中。三.我们将定期与斑马鱼国际资源中心（ZIRC）同步收集这些信息，以便长期存档并分发给斑马鱼社区。我们将使用最近建立的2D条形码库存系统和最先进的精子冷冻保存方法，以促进定期向ZIRC运送新品系。该建议是建立一个分子特征的集合，在硅片目录和分布网络的条件突变的杰出的非哺乳类脊椎动物，斑马鱼。这些显性标记的突变系也代表了作为最近启动的斑马鱼表型组项目的一部分的基因组的大规模行为和其他表型评估的理想底物。 公共卫生相关性：该提议是为斑马鱼（Danio rerio）产生2000个新的条件蛋白陷阱突变的突变体资源，斑马鱼是用于核心脊椎动物生物学研究和用于人类疾病建模的杰出的非哺乳动物系统。这些鱼线可通过马约诊所斑马鱼核心设施的分发实时获得。完整的收集将通过协调的国际努力建立，并由斑马鱼国际资源中心存档和分发，供全世界所有斑马鱼研究人员使用。

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.