Published online 2 August 2006
Nucleic Acids Research, 2006, Vol. 34, No. 13 e95
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Production of transmitochondrial cybrids containing naturally occurring pathogenic mtDNA variants
Mitochondrial Research Group, University of Newcastle upon Tyne, School of Neurology, Neurobiology and Psychiatry, The Medical School Framlington Place, Newcastle upon Tyne NE2 4HH, UK 1 Centre de Génétique Moléculaire, CNRS Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
*To whom correspondence should be addressed. Tel: +44 191 222 8028; Fax: +44 191 222 8553; Email: R.N.Lightowlers{at}ncl.ac.uk
Received April 21, 2006. Revised July 5, 2006. Accepted July 6, 2006.
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 transmitochondrial 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.
Present address: Deborah Pye, Division of Metabolic and Cellular Medicine, School of Clinical Sciences, University of Liverpool, Liverpool L69 3GA, UK
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Seibel, C. Di Nunno, C. Kukat, I. Schafer, R. Del Bo, A. Bordoni, G. P. Comi, A. Schon, F. Capuano, D. Latorre, et al. Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids Nucleic Acids Res., October 1, 2008; 36(18): 5872 - 5881. [Abstract] [Full Text] [PDF]
|
|