A procedure for selecting mammalian cells with an impairment in oxidative phosphorylation.
The mitochondrial encephalomyopathies in man are characterized by heterogeneous defects leading to an impairment in the pathway of aerobic energy production. As a means of investigating the molecular and genetic mechanisms underlying these disorders we have developed a procedure for selecting mammalian cell lines with features resembling the human pathological phenotypes. The principle of the selection is the use of a fluorescent amphiphilic dye, 2,4-(dimethylamino)-1-styrylmethylpyridiniumiodine, a cation showing two main features. Firstly, it is accumulated by mitochondria to an extent correlated with the magnitude of the electrochemical gradient of protons across the mitochondrial inner membrane. Secondly, upon irradiation with UV light, it gives rise to formation of free radicals, which inflict damage to the cell. Mutant cells with an impairment in oxidative phosphorylation will have more chance to survive than wild type cells. The selection procedure was applied to a stock of mutagenized Chinese hamster ovary cells. After subcloning of the cells which survived the selection procedure, twenty-six independent clones were isolated. Eighteen of the clones had a partial deficiency of cytochrome c oxidase ranging from 30 to 60% of the activity in control cells. The properties of two of the clones are described. One clone has been cultured under non-selective conditions for at least 12 months with retention of the partial deficiency of cytochrome c oxidase.
Lombardo
M C
MC
E.C. Slater Institute for Biochemical Research, University of Amsterdam, Academic Medical Centre, The Netherlands.
van der Zwaan
J W
JW
Brul
S
S
Tager
J M
JM
D000818:Animals; D019328:Buthionine Sulfoximine; D016466:CHO Cells / Q000201:enzymology*; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D030401:Cytochrome-c Oxidase Deficiency; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism; D004586:Electrophoresis; D006801:Humans; D015151:Immunoblotting; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000633:toxicity; D008928:Mitochondria / Q000201:enzymology*; D009154:Mutation; D010085:Oxidative Phosphorylation; D010641:Phenotype; D011726:Pyridinium Compounds / Q000494:pharmacology*; D014466:Ultraviolet Rays
D011726:Pyridinium Compounds; D008717:Methionine Sulfoximine; C011786:2-(4-(dimethylamino)styryl)-1-methylpyridinium; D019328:Buthionine Sulfoximine; D003576:Electron Transport Complex IV