Chromosome Engineering in Zebrafish

描述(申请人提供)：在后基因组时代，用功能信息注释人类基因组中所有已测序的基因是一个巨大的挑战，需要能够对基因和基因簇进行强大和高通量的正向和反向遗传分析的动物模型。斑马鱼(Danio Rerio)是一种小型淡水鱼，特别适合这种用途。它结合了无脊椎动物如果蝇(接受大规模突变和化学筛选)和哺乳动物如老鼠(比无脊椎动物模型更接近人类)的优点。斑马鱼具有体积小、外部发育、胚胎透明、易于维护和收集大量基因组信息等一系列独特特征，使其成为功能基因组学研究的极具吸引力的模型。然而，由于斑马鱼缺乏一些关键的遗传工具和资源，这一潜力并未完全实现。通过同源重组的基因打靶(敲除)是用于小鼠模型的最强大的遗传工具之一，但尚未在斑马鱼中开发。尽管已经在斑马鱼中建立了替代方法，但它们并不能产生真正的基因敲除。对主要由辐射产生的染色体重排突变体的鉴定已经显示出它们在研究基因功能、连锁座位的遗传相互作用和发现新基因方面的巨大作用。与这些辐射诱导的染色体重排相关的遗传问题，如绘制DNA损伤图谱和当缺失较大时为特定基因分配功能的困难，限制了它们在斑马鱼群落中的广泛应用。具有明确的和分子标记的染色体重排的大量突变体可以进一步操纵，是斑马鱼群落、功能基因组学和研究人类疾病不可或缺的资源。这项申请旨在为这些需要的资源开发一种新的方法。我们将斑马鱼中三种成熟的遗传工具整合到一个双转座子彩色报告系统中，以实现高效的基因打靶(敲除)和创造染色体重排。具体地说，我们利用睡美人(SB)和Tol2转座子的高效率进行随机染色体插入，并利用它们动员Cre/loxP系统进行定点重组的能力。我们使用荧光蛋白标记提供了非常高的灵敏度，便于检测和选择斑马鱼中的转座子移动和染色体重排。这一策略允许在预先选择的位置进行基因靶向(敲除)，并提供了比现有的辐射和化学诱变方法更多的优势。我们的方法和开发的试剂有几个独特的特点，使它们适用于单个斑马鱼实验室，并可以扩展到基因组水平。这项研究产生的突变将为研究这些突变基因在斑马鱼中的功能提供极好的资源，以更好地了解它们在人类和人类疾病中的作用。 公共卫生相关性：斑马鱼(Danio Rerio)是研究人类疾病的一种强大的模式生物。在这项建议中，我们将利用DNA转座子和Cre-loxP技术开发产生具有染色体重排的斑马鱼突变体的遗传方法。最终，这项工作可能会导致建立新的染色体工程技术和突变系的产生，这将为世界各地的科学家提供极好的资源，他们希望使用这项技术，研究这些突变基因在斑马鱼中的功能，以更好地了解它们在人类和人类疾病中的作用。

DESCRIPTION (provided by applicant): Annotating all the sequenced genes in the human genome with functional information is a great challenge in the postgenomic era and requires animal models that allow powerful and high throughput forward and reverse genetic analysis of genes and gene clusters. The zebrafish (Danio rerio), a small fresh water fish, is especially suitable for this purpose. It combines the advantages of both invertebrates such as Drosophila (subject to large-scale mutagenesis and chemical screening) and mammals such as mouse (more similar to humans than the invertebrate models). A number of unique features of the zebrafish such as small size, external development, embryonic transparency, easy maintenance, and a large collection of genomic information make it an incredibly attractive model for functional genomics studies. However, this potential is not fully realized due to the lack of some critical genetic tools and resources in zebrafish. Gene targeting (knockout) by homologous recombination, one of the most powerful genetic tools utilized in the mouse model, is yet to be developed in zebrafish. Although alternative methods have been established in zebrafish, they do not produce true knockouts. Characterization of chromosomal rearrangement mutants created primarily by radiation has already demonstrated their great utility in studying gene function, genetic interaction of linked loci, and discovery of new genes. Inherited problems associated with these radiation-induced chromosomal rearrangements, such as difficulties in mapping DNA lesions and assigning functions to a particular gene when a deletion is large, have limited their wide application in the zebrafish community. Large collections of mutants with defined and molecularly marked chromosomal rearrangements that can be further manipulated are indispensable resources for the zebrafish community, for functional genomics, and for studying human disease. This application aims to develop a novel method for these needed resources. We integrate three well- established genetic tools in zebrafish into a two-transposon color reporter system for efficient gene targeting (knockout) and creating chromosomal rearrangements. Specifically, we utilize the high efficiency of Sleeping Beauty (SB) and Tol2 transposons for random chromosomal insertion and their ability to mobilize, the power of Cre/loxP system for site-specific recombination. Our use of fluorescent protein markers provides very high sensitivity for easy detection and selection of transposon movement and chromosomal rearrangements in the zebrafish. This strategy permits gene targeting (knockout) at pre-selected sites and provides a number of advantages over the existing irradiation and chemical mutagenesis methods. Several unique features of our methods and developed reagents make them applicable to individual zebrafish laboratories and can be scaled to genome levels. Mutants generated in this study will provide excellent resources for studying the function of these mutated genes in the zebrafish to better understand their role in humans and human diseases. PUBLIC HEALTH RELEVANCE: The zebrafish (Danio rerio) is a powerful model organism for studying human diseases. In this proposal, we will develop genetic methods for generation of zebrafish mutants with chromosomal rearrangements by using DNA transposons and the Cre-loxP technology. Ultimately, this work may lead to establishment of novel chromosome engineering technology and generation of mutant lines, which will provide excellent resources for scientists worldwide who wish to use this technology and study the function of these mutated genes in the zebrafish to better understand their role in humans and human diseases.