International Zebrafish Mutagenic Protein Trap

描述(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模拟人类疾病的卓越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、苍蝇和老鼠领域的强大工具。基因组学方法已经很好地表征了核基因组和转录组。然而，在动态背景下和体内对蛋白质组全部复杂性的基因组评估仍在很大程度上 未知。斑马鱼使用整个脊椎动物基因组作为模板，提供了第一个全面的蛋白质组分析。我们发现，突变蛋白陷阱基因断裂转座子(GBTS)是探索发育生物学(包括器官发生)等传统领域的有效和可逆的功能丧失工具，但也开辟了包括行为遗传学在内的新选择，如尼古丁反应生物学。一组Cre重组酶可调节的斑马鱼突变等位基因提供了在组织、器官和细胞水平上探索脊椎动物生物学关键问题的机会。这项申请是对“PAR-08-139利用研究工具和技术(R01)加强斑马鱼研究”的响应，目的是通过协调国际努力，完成斑马鱼群落这些可逆转突变等位基因大规模收集的进展。我们将在项目结束时提供以下内容：I.将建立1000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zFish book.org实时提供标签表达模式的基本描述。这些产品立即可以通过梅奥诊所斑马鱼核心设施的分销获得。II.我们将对这些线进行分子分析，并随后对斑马鱼基因组计划进行注释，以确定该标本中捕获的蛋白质。我们将继续使用我们成功的下一代序列技术和连锁分析来分离和定位斑马鱼基因组上的GBT插入。所有序列信息最初将通过zFish book.org分发，然后整合到ZFIN、FishMap和其他斑马鱼基因组数据库中。我们将定期与斑马鱼国际资源中心(ZIRC)同步这些收藏，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运送新的生产线。这项建议是为了完成这个分子特征的收集，在电子目录和分布网络的可逆突变的卓越的非哺乳动物脊椎动物，斑马鱼。这些主要标记的突变系也是大规模行为和其他表型基因组评估的理想底物，作为使用斑马鱼对脊椎动物基因组进行功能注释的更广泛倡议的一部分。

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 revertible 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. A panel of Cre recombinase regulatable mutant zebrafish alleles offers the opportunity to explore critical questions in vertebrate biology at the tissue, organ and cellular level of resolution. This application is in response to "PAR-08-139 Enhancing Zebrafish Research with Research Tools and Techniques (R01)" to complete progress on the development of a large-scale collection of these revertible mutant alleles for the zebrafish community through a coordinated international effort. We will deliver the following at project's end: I. 1000 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 continue to use our successful next generation sequence techniques followed by linkage analysis to isolate and map GBT insertions on the zebrafish genome. 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 complete this molecularly characterized collection, in silico catalog and distribution network of revertible 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 broader initiative to functionally annotate the vertebrate genome using the zebrafish.