Generation of ρ0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Eukaryotic cells devoid of mitochondrial DNA (ρ⁰ 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. ρ⁰ 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 ρ⁰ cell lines, we developed an extremely mild, reliable and timesaving method to generate ρ⁰ 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 ρ⁰ cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK⁻ ρ⁰ cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human ρ⁰ mitochondria.