title: Methods for Efficient Elimination of Mitochondrial DNA from Cultured Cells.
abstract: Here, we document that persistent mitochondria DNA (mtDNA) damage due to mitochondrial overexpression of the Y147A mutant uracil-N-glycosylase as well as mitochondrial overexpression of bacterial Exonuclease III or Herpes Simplex Virus protein UL12.5M185 can induce a complete loss of mtDNA (ρ0 phenotype) without compromising the viability of cells cultured in media supplemented with uridine and pyruvate. Furthermore, we use these observations to develop rapid, sequence-independent methods for the elimination of mtDNA, and demonstrate utility of these methods for generating ρ0 cells of human, mouse and rat origin. We also demonstrate that ρ0 cells generated by each of these three methods can serve as recipients of mtDNA in fusions with enucleated cells.
authors.affiliation: Department of Pharmacology, University of South Alabama, Mobile, Alabama, United States of America.
authors.forename: Domenico
authors.initials: D
authors.lastname: Spadafora
authors.affiliation: Department of Physiology and Cell Biology, University of South Alabama, Mobile, Alabama, United States of America.
authors.forename: Nataliya
authors.initials: N
authors.lastname: Kozhukhar
authors.affiliation: Division of Biotechnology & Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Chouljenko
authors.affiliation: Division of Biotechnology & Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Kousoulas
authors.affiliation: Department of Physiology and Cell Biology, University of South Alabama, Mobile, Alabama, United States of America.|Center for Lung Biology, University of South Alabama, Mobile, Alabama, United States of America.
authors.forename: Mikhail F
authors.initials: MF
authors.lastname: Alexeyev
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D004249:DNA Damage / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000235:genetics / Q000502:physiology; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D017382:Reactive Oxygen Species