Abstract
In farm animals, mitochondrial DNA mutations exist widely across breeds and individuals. In order to identify differences among mtDNA haplotypes, two porcine transmitochondrial cybrids were generated by fusion of a Lantang pig cell line devoid of mitochondrial DNA with enucleated cytoplasm from either a Large White pig or a Xiang pig harboring potentially divergent mitochondrial haplotypes. These cybrid cells were subjected to mitochondrial genome sequencing, copy number detecting and analysis of biochemical traits including succinate dehydrogenase (SDH) activity, ATP content and susceptibility to reactive oxygen species (ROS). The Lantang and Xiang mitochondrial genomes were highly homologous with only 18 polymorphic sites, and differed radically from the Large White with 201 and 198 mutations respectively. The Large White and Xiang cybrids exhibited similar mtDNA copy numbers and different values among biochemical traits, generated greater ROS production (P < 0.05) and less SDH activity (P < 0.05) and a lesser ATP content (P < 0.05). The results show that functional differences exist between cybrid cells which differ in mitochondrial genomic background. In conclusion, transmitochondrial cybrids provide the first direct evidence on pig biochemical traits linking different mitochondrial genome haplotypes.
Highlights
In farm animals, mtDNA variations were reported in association with complex traits in pigs[4,5], cattle[6,7,8], chickens[9], ducks[10] and donkeys[11]
In order to introduce selective markers for recipient cells devoid of mitochondrial DNA (ρ 0 cells), a GFP-neo gene was introduced into C1 cell line, with a G418 selectable marker recognized under fluorescence (Fig. 2,a1,a2)
Confirmation was obtained by Janus green B (JG-B) staining which labels vital mitochondria by reduction and oxidation of the electron transfer chain alteration[16]
Summary
MtDNA variations were reported in association with complex traits in pigs[4,5], cattle[6,7,8], chickens[9], ducks[10] and donkeys[11]. The phenotypic effects of variations in the mitochondrial genome were difficult to isolate owing to confounding variation in the nuclear genome, epigenetic phenomena, and environmental factors. Few animal models have been available for directly investigating the effect of mtDNA variations on complex metabolic phenotypes in vivo until the creation of transmitochondrial cybrids[12]. We isolated somatic cells from three pig breeds representing different mitochondrial genome haplotypes: Lantang (C0), Xiang (C1) and Large White (C2). Their mitochondrial DNAs and variations were characterized. The model would allow separation of phenotypic changes resulting from mitochondrial genome variation from confounding influences imparted by nuclear gene expression, epigenetic phenomena and environmental factors. This study provides the first evidence detailing the mtDNA effect on targeted biochemical traits in pig cells
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