Mutagenesis of mtDNA in Zebrafish

Dartmouth College, Hanover NH

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Abstract

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.