Generation of ρ 0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Abstract

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.

Involvement of p32 and Microtubules in Alteration of Mitochondrial Functions by Rubella Virus

“…2 and 3). Therefore, the contribution of mitochondrial activity to the RV life cycle was investigated through infection of a cell line that was completely depleted of mitochondrial DNA (26). The so-called rho 0 derivative of the 143B.TK Ϫ osteosarcoma cell line was generated by a mild enzymatic approach and lacks a functional respiratory chain.…”

“…The so-called rho 0 derivative of the 143B.TK Ϫ osteosarcoma cell line was generated by a mild enzymatic approach and lacks a functional respiratory chain. It serves as a model for analysis of the importance of oxidative phosphorylation (oxphos) for cellular processes (26,35). The 143B.TK Ϫ cell line enables a productive infection with RV and yields virus titers comparable to those of Vero cells (Fig.…”

“…Vero (ATCC CCL-81), BHK21 (ATCC CCL-10), HEK293 (ATCC CRL-1573), and HEK293T/17 (ATCC CRL-11268) cell lines and the human osteosarcoma-derived cell line 143B.TK Ϫ and its mitochondrial-DNA-depleted derivate 143B.TK Ϫ rho 0 (26) were maintained in Dulbecco's modified Eagle's medium (DMEM) with high glucose (PAA, Cölbe, Germany). Medium for the 143B.TK Ϫ and 143B.TK Ϫ rho 0 cell lines was supplemented with 50 g/ml uridine (to compensate for a blockade in the pyrimidine biosynthetic pathway) and 1 mM sodium pyruvate (a hydrogen acceptor to regenerate NAD from NADH to support energy generation by glycolysis).…”

Depletion of mitochondria in mammalian cells through enforced mitophagy

“…Alternative methods to deplete mtDNA and generate ρ 0 cells include mitochondrial targeting of the restriction endonuclease EcoRI 18 or expression of a dominant negative form of Polγ, a gene that codes for the catalytic subunit of the mitochondrial DNA polymerase 19 . However, these solutions do not circumvent a major drawback, which is that ρ 0 cells still contain functional mitochondria.…”

An Impaired Respiratory Electron Chain Triggers Down-regulation of the Energy Metabolism and De-ubiquitination of Solute Carrier Amino Acid Transporters

“…Human cells lacking mtDNA ( 0 cells) were originally obtained from the human osteosarcoma cell line 143B.TK Ϫ (7) by chronic exposure to the DNA intercalating dye ethidium bromide. Since then, 0 cell lines have been established from various tissues and species applying additional methods such as application of the anticancer drug ditercalinium (8) or restriction enzymes specifically targeting mitochondria (9,10). However, 0 cells are viable in culture, provided appropriate conditions are met (11), e.g.…”