TARGETED MUTAGENESIS IN ZEBRAFISH BY ANIMAL CLONING

描述(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。几个大规模的突变筛选已经产生了数千条在重要生物过程中存在缺陷的突变鱼。然而，该系统仍然缺乏进行反向遗传学和基因标签突变的有效方法。最近，我们已经证明，利用长期培养的细胞的供体核，可以通过核移植来克隆正常的斑马鱼个体。在这项提案中，我们将在两个领域探索这一发现的实用性。首先，我们将建立一个基因捕获程序，通过该程序，培养的细胞将被选择用于基因捕获事件，然后用于通过动物克隆来生产斑马鱼。第二，我们将开发斑马鱼反向遗传学的定向突变方法。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打乱，携带同源重组事件的细胞核将被用于生产克隆斑马鱼。在过去的三年里，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打靶技术。聚集了一批具有独特技术才华的科学家。在持续不断的支持下，我们相信我们可以实现拟议的目标，斑马鱼很快就会提供老鼠研究界所享受的所有基本工具。这两个系统将真正相辅相成，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.