Mutagenesis of mtDNA in Zebrafish

斑马鱼线粒体DNA的诱变

• 批准号: 6547092
• 负责人: Glenn S Gerhard
• 金额: $ 6.5万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6547092
• 关键词: DNA damage; aging; alternatives to animals in research; biotechnology; electron transport; embryo /fetus; fluoresceins; free radical oxygen; gamma radiation; gene deletion mutation; genetic manipulation; genetic models; genetic screening; mitochondrial DNA; model design /development; mutagens; radiation dosage; temperature; zebrafish

The long term goals of this proposal are to create zebrafish harboring mitochondrial DNA (mtDNA) deletions, and to identify mutants withy altered levels of (ROS). Deletions in mtDNA increase with age in multiple tissues and multiple species and may be caused by reactive oxygen species (ROS). The development of mouse models harboring mtDNA mutations have been technically challenging, and, when successful, phenotypically divergent from humans. Large-scale mutagenesis techniques have been used to identify thousands of zebrafish mutants with developmental defects, but current screening approaches generally neglect the mitochondrial genome. Our hypothesis is that gamma radiation will induce deletions in the mtDNA of germ cells of early stage zebrafish embryos, and that mutations in either nuclear or mitochondrial genes will occur that will affect the production of ROS. Our rationale is that irradiation has been reported to induce deletions in the nuclear DNA of zebrafish and in the mtDNA of cultured cell lines. The optimum dosage and schedule of irradiation required to induce mtDNA deletions while maintaining viability, normal development, and fertility will be determined. Fish derived from mutagenized embryos will be screened for mtDNA deletions using PCR based assays and bred to normal fish to segregate mtDNA. Several adjuvant strategies to facilitate the formation of mtDNA deletions will also be tested in conjunction with irradiation, including temperature modulation, increasing ROS production with inhibitors of electron transport and pro-oxidants, and alteration of mtDNA content. Mutagenized fish will also be screened using a fluoroscein compound to detect in vivo ROS generation. Our specific aims are to 1) Establish dose and survival parameters for radiation mutagenesis in early stage zebrafish embryos; 2) identify and characterize induced mtDNA mutations; 3) Screen mutagenized zebrafish for in vivo ROS production using dichlorodihydrofluorescein diacetate (DCF) Zebrafish with mtDNA deletions could serve as animal models for both human mtDNA disorders, and for studying the role of low level mtDNA mutations in aging. The identification of mutants that manifest altered ROS production will be of particular interest to investigate the role that ROS generation plays in aging and longevity.

该提案的长期目标是培育含有线粒体 DNA (mtDNA) 缺失的斑马鱼，并鉴定 (ROS) 水平发生改变的突变体。在多个组织和多个物种中，mtDNA 的缺失随着年龄的增长而增加，并且可能是由活性氧 (ROS) 引起的。携带 mtDNA 突变的小鼠模型的开发在技术上具有挑战性，而且一旦成功，其表型就会与人类不同。大规模诱变技术已被用来识别数千种具有发育缺陷的斑马鱼突变体，但目前的筛选方法通常忽略线粒体基因组。我们的假设是，伽马辐射会诱导早期斑马鱼胚胎生殖细胞线粒体 DNA 缺失，并且核或线粒体基因会发生突变，从而影响 ROS 的产生。我们的理由是，据报道，辐射会引起斑马鱼核 DNA 和培养细胞系 mtDNA 的缺失。将确定诱导 mtDNA 缺失同时维持活力、正常发育和生育能力所需的最佳照射剂量和时间表。来自诱变胚胎的鱼将使用基于 PCR 的检测来筛选 mtDNA 缺失，并与正常鱼繁殖以分离 mtDNA。一些促进 mtDNA 缺失形成的辅助策略也将与辐射结合进行测试，包括温度调节、使用电子传递抑制剂和促氧化剂增加 ROS 的产生，以及改变 mtDNA 含量。诱变鱼也将使用荧光素化合物进行筛选，以检测体内 ROS 的产生。我们的具体目标是 1) 建立早期斑马鱼胚胎辐射诱变的剂量和存活参数； 2) 识别和表征诱导的 mtDNA 突变； 3) 使用二氯二氢荧光素二乙酸酯 (DCF) 筛选诱变斑马鱼以产生体内 ROS。 mtDNA 缺失的斑马鱼可以作为人类 mtDNA 疾病和研究低水平 mtDNA 突变在衰老中的作用的动物模型。鉴定表现出 ROS 产生改变的突变体对于研究 ROS 产生在衰老和长寿中所起的作用特别有意义。