Mutagenesis of mtDNA in Zebrafish

该提案的长期目标是创造具有线粒体DNA （mtDNA）缺失的斑马鱼，并识别具有改变（ROS）水平的突变体。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，可能是由活性氧（ROS）引起的。开发携带mtDNA突变的小鼠模型在技术上具有挑战性，并且，当成功时，表型上与人类不同。大规模诱变技术已被用于鉴定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了线粒体基因组。我们的假设是，伽马辐射会导致早期斑马鱼胚胎生殖细胞mtDNA的缺失，并且会发生核或线粒体基因的突变，从而影响ROS的产生。我们的基本原理是，据报道辐照会导致斑马鱼核DNA和培养细胞系mtDNA的缺失。将确定诱导mtDNA缺失同时保持生存能力、正常发育和生育能力所需的最佳照射剂量和时间表。从诱变胚胎中获得的鱼将使用基于PCR的检测方法筛选mtDNA缺失，并与正常鱼繁殖以分离mtDNA。几种促进mtDNA缺失形成的辅助策略也将与辐照结合进行测试，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mtDNA含量的改变。诱变鱼也将使用氟蛋白化合物进行筛选，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)鉴定和表征诱导的mtDNA突变；3)利用二氯二氢荧光素（DCF）筛选诱变斑马鱼体内ROS的产生。mtDNA缺失的斑马鱼可以作为人类mtDNA疾病的动物模型，也可以用于研究低水平mtDNA突变在衰老中的作用。识别显示ROS生成改变的突变体将对研究ROS生成在衰老和长寿中所起的作用特别感兴趣。

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.