Genome Instability in Cells and Tissues of the Zebrafish

斑马鱼细胞和组织的基因组不稳定性

• 批准号: 7140162
• 负责人: FORREST A. SPENCER
• 金额: $ 15.97万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-26 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140162
• 关键词: biotechnology; gene induction /repression; gene mutation; genetic markers; genetic screening; genome; green fluorescent proteins; neoplasm /cancer genetics; neoplastic growth; technology /technique development; zebrafish

DESCRIPTION (provided by applicant): Change in genome structure can occur in mitotic and meiotic cell lineages, and this contributes to individual variation, evolution, and disease. It has been argued that stochastic somatic genome instability makes an early and important contribution to the development of human cancer, largely through loss of wild-type tumor suppressor genes. Many tumor suppressor genes themselves are guardians of genome structure and proper cell cycle control, and their loss may cause additional somatic instability. Thus, high levels of genomic instability may be viewed as possible cause and/or effect of steps in tumorigenesis. To distinguish these, a method is needed for continuous monitoring of genome stability in cell lineages that give rise to cancer. Genome stability in vertebrates is currently followed using karyotype analysis, fluorescence in situ hybridization, or measurement of endogenous marker loss using cell selection procedures. At this time, an in vivo system that can be used to determine genome stability in situ (i.e., without tissue disruption) is lacking. We propose the zebrafish Danio rerio as an ideal model system in which to develop this view of vertebrate biology, and we outline a novel method for following marker stability in fish. The method is based on a transcriptional repression design in which repressor loss leads to expression of the fluorescent protein EGFP. Using transgenic zebrafish, we will perform proof-of-concept tests for detection of repressor loss and characterization of repressor loss mechanisms. The zebrafish is ideally suited for development of this strategy due to the ease of organ visualization and its well-developed genetics and genomics tools. Furthermore, the rapid generation time and small size of the zebrafish supports cost-effective observation of many individuals, providing statistical power. In future work, this measurement of genome stability in situ will be important for understanding the relationship between gene function and genome instability in different tissues, and between genome instability and tumor development.

描述（由申请人提供）：基因组结构的变化可能发生在有丝分裂和减数分裂细胞系中，这有助于个体变异、进化和疾病。人们一直认为，随机体细胞基因组不稳定性对人类癌症的发展起着早期和重要的作用，主要是通过野生型肿瘤抑制基因的丧失。许多肿瘤抑制基因本身是基因组结构和适当细胞周期控制的守护者，它们的丢失可能导致额外的体细胞不稳定。因此，高水平的基因组不稳定性可能被视为肿瘤发生步骤的可能原因和/或结果。为了区分这些，需要一种方法来连续监测导致癌症的细胞系的基因组稳定性。目前使用核型分析、荧光原位杂交或使用细胞选择程序测量内源性标记损失来跟踪脊椎动物的基因组稳定性。此时，缺乏可用于确定原位基因组稳定性（即不破坏组织）的体内系统。我们提出斑马鱼Danio rerio作为发展这种脊椎动物生物学观点的理想模型系统，并概述了一种跟踪鱼类标记稳定性的新方法。该方法基于转录抑制设计，其中抑制因子丢失导致荧光蛋白EGFP的表达。使用转基因斑马鱼，我们将进行概念验证测试，以检测抑制因子损失和表征抑制因子损失机制。由于器官可视化的便利性及其发达的遗传学和基因组学工具，斑马鱼非常适合这种策略的发展。此外，斑马鱼的快速生成时间和小尺寸支持对许多个体进行经济有效的观察，提供了统计能力。在未来的工作中，这种原位基因组稳定性的测量对于理解不同组织中基因功能与基因组不稳定性之间的关系以及基因组不稳定性与肿瘤发展之间的关系将是重要的。