Chromosome Engineering in Zebrafish

描述（由申请人提供）：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项巨大的挑战，这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），一种小型淡水鱼，特别适合这个目的。它结合了果蝇等无脊椎动物（需要进行大规模诱变和化学筛选）和老鼠等哺乳动物（与无脊椎动物模型相比，与人类更相似）的优点。斑马鱼的一些独特特征，如体积小，外部发育，胚胎透明，易于维护，以及大量的基因组信息收集，使其成为功能基因组学研究的一个非常有吸引力的模型。然而，由于缺乏一些关键的遗传工具和资源，斑马鱼的这种潜力并没有完全实现。同源重组的基因靶向（敲除）是小鼠模型中最强大的遗传工具之一，但在斑马鱼中尚未开发。尽管在斑马鱼中已经建立了替代方法，但它们并不能产生真正的击倒。主要由辐射产生的染色体重排突变的特征已经证明了它们在研究基因功能、连锁位点的遗传相互作用和发现新基因方面的巨大效用。与这些辐射诱导的染色体重排相关的遗传问题，如DNA损伤定位和当缺失较大时分配特定基因功能的困难，限制了它们在斑马鱼群落中的广泛应用。对于斑马鱼群落、功能基因组学和人类疾病研究来说，大量具有明确和分子标记的染色体重排的突变体是不可缺少的资源。本应用程序旨在为这些所需资源开发一种新的方法。我们将三种完善的遗传工具整合到斑马鱼的双转座子颜色报告系统中，用于有效的基因靶向（敲除）和创建染色体重排。具体来说，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和它们的动员能力，以及Cre/loxP系统进行位点特异性重组的能力。我们使用荧光蛋白标记为斑马鱼转座子运动和染色体重排的检测和选择提供了非常高的灵敏度。这种策略允许在预先选择的位点进行基因靶向（敲除），并且比现有的辐照和化学诱变方法提供了许多优势。我们的方法和开发的试剂的几个独特功能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。本研究产生的突变体将为研究这些突变基因在斑马鱼中的功能，更好地了解它们在人类和人类疾病中的作用提供极好的资源。

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.