Generation of Rho Zero Cells: Visualization and Quantification of the mtDNA Depletion Process.
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.
Molecular Cell Therapy, Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, 04103 Leipzig, Germany. susanna.schubert@bbz.uni-leipzig.de.|Translational Centre for Regenerative Medicine (TRM), Universität Leipzig, 04103 Leipzig, Germany. susanna.schubert@bbz.uni-leipzig.de.
Susanna
S
Schubert
Molecular Cell Therapy, Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, 04103 Leipzig, Germany. sheller@tulane.edu.|Department of Pathology and Laboratory Medicine, Tulane University, New Orleans, LA 70112, USA. sheller@tulane.edu.
Sandra
S
Heller
Molecular Cell Therapy, Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, 04103 Leipzig, Germany. birgit.loeffler@bbz.uni-leipzig.de.
Birgit
B
Löffler
Molecular Cell Therapy, Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, 04103 Leipzig, Germany. ingo.schaefer@bbz.uni-leipzig.de.
Ingo
I
Schäfer
RhoZero Technologies, 97292 Uettingen, Germany. martina.seibel@rhozero.com.
Martina
M
Seibel
Molecular Cell Therapy, Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, 04103 Leipzig, Germany. gaetano.villani@uniba.it.|Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari, 70124 Bari, Italy. gaetano.villani@uniba.it.
Gaetano
G
Villani
Molecular Cell Therapy, Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, 04103 Leipzig, Germany. peter.seibel@bbz.uni-leipzig.de.
Peter
P
Seibel
D045744:Cell Line, Tumor; D004272:DNA, Mitochondrial / Q000378:metabolism*; D054629:Genome, Mitochondrial; D006801:Humans; D013937:Thymidine Kinase / Q000378:metabolism; D014162:Transfection
D004272:DNA, Mitochondrial; D013937:Thymidine Kinase