Generation of Rho Zero Cells: Visualization and Quantification of the mtDNA Depletion Process.

ABSTRACT: 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

SUBMITTER: Schubert S 

PROVIDER: S-EPMC4463621 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature