Nucleic Acids Research Advance Access originally published online on March 19, 2008
Nucleic Acids Research 2008 36(7):e44; doi:10.1093/nar/gkn124
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Nucleic Acids Research, 2008, Vol. 36, No. 7 e44
© 2008 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-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Generation of 
0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses
1Molecular Cell Therapy, Center for Biotechnology and Biomedicine, Faculty of Medicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany, 2Karolinska Institutet, Department of Laboratory Medicine, Division of Metabolic Diseases, Novum, 141 86 Stockholm, Sweden, 3Department of Cell and Developmental Biology, 4Division of Electron Microscopy, Biocenter of the University of Würzburg, Am Hubland, 97074 Würzburg, Germany, 5Centre for Clinical Neurosciences, The University of Melbourne Department of Medicine, St Vincent's Hospital, Fitzroy, Victoria 3065, Australia and 6Department of Medical Biochemistry, Biology & Physics, University of Bari, Piazza Giulio Cesare, 11, 70124 Bari, Italy
*To whom correspondence should be addressed. Tel: +49 341 9731370; Fax: +49 341 9731379; Email: peter.seibel{at}bbz.uni-leipzig.de
Received December 13, 2007. Revised March 3, 2008. Accepted March 4, 2008.
Eukaryotic cells devoid of mitochondrial DNA (
0 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. 0 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 0 cell lines, we developed an extremely mild, reliable and timesaving method to generate 0 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 0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK– 0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human 0 mitochondria.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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