Generation of rho0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses.
Eukaryotic cells devoid of mitochondrial DNA (rho0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. Rho0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of rho0 cell lines, we developed an extremely mild, reliable and timesaving method to generate rho0 cell lines within 3-5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK- the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat rho0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK- rho0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human rho0 mitochondria.
Kukat
Alexandra
A
Molecular Cell Therapy, Center for Biotechnology and Biomedicine, Faculty of Medicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
Kukat
Christian
C
Brocher
Jan
J
Schäfer
Ingo
I
Krohne
Georg
G
Trounce
Ian A
IA
Villani
Gaetano
G
Seibel
Peter
P
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D003470:Culture Media; D004272:DNA, Mitochondrial / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000235:genetics* / Q000378:metabolism; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D021382:Protein Sorting Signals; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D003470:Culture Media; D004272:DNA, Mitochondrial; D005456:Fluorescent Dyes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015246:Deoxyribonuclease EcoRI