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Human Molecular Genetics, 1999, Vol. 8, No. 9 1691-1697
© 1999 Oxford University Press

A novel system for assigning the mode of inheritance in mitochondrial disorders using cybrids and rhodamine 6G

Andrew J. Williams1,2, Melanie Murrell2, Susan Brammah3, Jim Minchenko2 and John Christodoulou2,4,+

1Department of Clinical Biochemistry and 4Western Sydney Genetics Program, Royal Alexandra Hospital for Children, Hawkesbury Road, Westmead, Australia, 2Department of Paediatrics and Child Health, University of Sydney, Sydney, Australia and 3Electron Microscopy Unit, Central Sydney Area Health Service, Concord, Australia

When normal human cultured skin fibroblasts were treated with the fluorescent dye rhodamine 6G (R6G), there was a drastic reduction in numbers of intact mitochondria and electron transport chain enzyme activities, despite the fact that mitochondrial DNA (mtDNA) was still present in treated cells. We used this observation to develop a novel system for generating cybrids. When cultured skin fibroblast cells from a patient with the mitochondrial encephalopathy and ragged-red fibers (MERRF) syndrome harboring the A8344G mtDNA mutation and which showed a severe reduction in cytochrome c oxidase activity were treated with R6G and fused to enucleated HeLaCOT cells, the resulting cybrid clones showed recovery of cytochrome c oxidase activity, and were shown to have mtDNA derived solely from the HeLaCOT cell line. R6G has significant advantages over ethidium bromide in removing the mitochondrial elements from cultured cells, and the results reported here demonstrate that this strategy can be used to determine the origin of the genetic defect in patients with electron transport chain abnormalities.

+ To whom correspondence should be addressed at: Western Sydney Genetics Program, Royal Alexandra Hospital for Children, Hawkesbury Road, Westmead, NSW 2145, Australia. Tel: +61 2 9845 3452; Fax: +61 2 9845 1864; Email: johnch{at}mail.usyd.edu.au


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