TARGETED MUTAGENESIS IN ZEBRAFISH BY ANIMAL CLONING

描述（由申请人提供）：斑马鱼是研究脊椎动物发育和模拟人类疾病的极好的遗传系统。几次大规模的诱变筛选已经产生了数千种在重要生物过程中存在缺陷的突变鱼。然而，该系统仍然缺乏有效的方法来执行反向遗传和基因标记诱变。最近，我们已经证明正常的斑马鱼个体可以通过核移植使用长期培养细胞的供体细胞核进行克隆。在本提案中，我们将在两个方面探讨这一发现的效用。首先，我们将建立一个基因诱捕程序，通过该程序将选择培养的细胞进行基因诱捕事件，然后通过动物克隆用于生产斑马鱼。其次，我们将开发斑马鱼反向遗传的靶向诱变方法。在培养的成纤维细胞中，一些选定的候选基因将被同源重组破坏，然后携带同源重组事件的细胞核将被用于克隆斑马鱼。在过去的三年中，我们的实验室投入了大量的资源和时间来开发斑马鱼的基因靶向技术。聚集了一批具有独特技术才能的科学家。在持续的支持下，我们相信我们可以实现所提出的目标，斑马鱼将很快提供所有小鼠研究界所享受的基本工具。这两个系统将真正互补，以促进我们对脊椎动物发育生物学的理解。

DESCRIPTION (provided by applicant): The zebrafish is a superb genetic system for studying vertebrate development and modeling human diseases. Several large-scale mutagenesis screens have generated thousands of mutant fish with defects in important biological processes. However, this system still lacks efficient ways to perform reverse genetics and gene tagged mutagenesis. Recently, we have shown that normal zebrafish individuals can be cloned by nuclear transfer using donor nuclei from long-term cultured cells. In this proposal, we will explore the utility of this finding in two areas. First, we will establish a gene trap procedure by which cultured cells will be selected for gene trap events and then used to produce zebrafish by animal cloning. Second, we will develop targeted mutagenesis methods for reverse genetics in zebrafish. A number of selected candidate genes will be disrupted by homologous recombination in cultured fibroblast cells and the nuclei carrying the homologous recombination events will then be used to produce cloned zebrafish. For the past three years, our laboratory has devoted significant resources and time towards developing gene-targeting technology in zebrafish. A group of scientists with unique technological talents have been assembled. With a continuous support, we believe that we can achieve the proposed goals and zebrafish will soon offer all the essential tools that have been enjoyed by the mouse research community. The two systems will then truly complement each other to advance our understanding of vertebrate developmental biology.