title: Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker.
abstract: Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.
authors.affiliation: Mitochondria Hub Regulation Center and Department of Anatomy and Cell Biology, Dong-A University College of Medicine, 3-1 Dongdaesin-dong, Seo-gu, Busan 602-714, Republic of Korea. youngyoon97@gmail.com
authors.forename: Young Geol
authors.initials: YG
authors.lastname: Yoon
authors.forename: Michael Duane
authors.initials: MD
authors.lastname: Koob
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance / Q000187:drug effects / Q000235:genetics*; D023061:Gene Order; D005839:Gentamicins / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D014162:Transfection
chemical_list: D004272:DNA, Mitochondrial; D005839:Gentamicins; C010680:antibiotic G 418; D019868:Kanamycin Kinase