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Production of transmitochondrial cybrids containing naturally occurring pathogenic mtDNA variants

Lookup NU author(s): Dr Debbie Pye, Dimitra-Smaragda Kyriakouli, Geoffrey Taylor, Dr Matthias Elstner, Professor Zofia Chrzanowska-LightowlersORCiD, Professor Robert Taylor, Emeritus Professor Doug Turnbull, Professor Robert Lightowlers

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Abstract

The human mitochondrial genome (mtDNA) encodes polypeptides that are critical for coupling oxidative phosphorylation. Our detailed understanding of the molecular processes that mediate mitochondrial gene expression and the structure-function relationships of the OXPHOS components could be greatly improved if we were able to transfect mitochondria and manipulate mtDNA in vivo. Increasing our knowledge of this process is not merely of fundamental importance, as mutations of the mitochondrial genome are known to cause a spectrum of clinical disorders and have been implicated in more common neurodegenerative disease and the ageing process. In organellar or in vitro reconstitution studies have identified many factors central to the mechanisms of mitochondrial gene expression, but being able to investigate the molecular aetiology of a limited number of cell lines from patients harbouring mutated mtDNA has been enormously beneficial. In the absence of a mechanism for manipulating mtDNA, a much larger pool of pathogenic mtDNA mutations would increase our knowledge of mitochondrial gene expression. Colonic crypts from ageing individuals harbour mutated mtDNA. Here we show that by generating cytoplasts from colonocytes, standard fusion techniques can be used to transfer mtDNA into rapidly dividing immortalized cells and, thereby, respiratory-deficient trans mitochondrial cybrids can be isolated. A simple screen identified clones that carried putative pathogenic mutations in MTRNR1, MTRNR2, MTCOI and MTND2, MTND4 and MTND6. This method can therefore be exploited to produce a library of cell lines carrying pathogenic human mtDNA for further study. © Copyright 2006 Oxford University Press.

Publication metadata

Author(s): Pye D, Kyriakouli DS, Taylor GA, Johnson R, Elstner M, Meunier B, Chrzanowska-Lightowlers ZMA, Taylor RW, Turnbull DM, Lightowlers RN

Publication type: Article

Publication status: Published

Journal: Nucleic Acids Research

Year: 2006

Volume: 34

Issue: 13

ISSN (print): 0305-1048

ISSN (electronic): 1362-4962

Publisher: Oxford University Press

URL: http://dx.doi.org/10.1093/nar/gkl516

DOI: 10.1093/nar/gkl516

PubMed id: 16885236

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