Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein
complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases.
By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during
this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:bsz:15-qucosa-167888 |
| Date | 30 April 2015 |
| Creators | Schubert, Susanne, Heller, Sandra, Löffler, Birgit, Schäfer, Ingo, Seibel, Martina, Villani, Gaetano, Seibel, Peter |
| Contributors | Universität Leipzig, Biotechnologisch-Biomedizinische Zentrum, Universität Leipzig, Translationszentrum für Regenerative Medizin, Tulane University, Department of Pathology and Laboratory Medicine, University of Bari, Department of Basic Medical Sciences, Neurosciences and Sense Organs, RhoZero Technologies,, Universität Leipzig, |
| Publisher | Universitätsbibliothek Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:article |
| Format | application/pdf |
| Source | International Journal of Molecular Sciences 2015 : 16, 9850-9865 |
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