title: Inducible expression of a dominant negative DNA polymerase-gamma depletes mitochondrial DNA and produces a rho0 phenotype.
abstract: We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-gamma to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-gamma harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in rho0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the rho0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-gamma can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation.
authors.affiliation: MitoKor, Inc., San Diego, California 92121, USA.
authors.forename: Mona
authors.initials: M
authors.lastname: Jazayeri
authors.forename: Alexander
authors.initials: A
authors.lastname: Andreyev
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Will
authors.forename: Manus
authors.initials: M
authors.lastname: Ward
authors.forename: Christen M
authors.initials: CM
authors.lastname: Anderson
authors.forename: William
authors.initials: W
authors.lastname: Clevenger
mesh_terms: D000409:Alanine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D002455:Cell Division; D002459:Cell Fusion; D002460:Cell Line; D000074002:DNA Polymerase gamma; D018076:DNA, Complementary / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005799:Genes, Dominant; D005822:Genetic Vectors; D006801:Humans; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D012333:RNA, Messenger; D011994:Recombinant Proteins; D001224:Aspartic Acid; D012313:RNA; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D000409:Alanine