Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining.
Human mitochondria DNA (mtDNA) is arranged within the mitochondria into discrete DNA-protein complexes, termed nucleoids. The size of the human mitochondrial genome is less than that of yeast and is more difficult to visualise by fluorescent DNA stains such as DAPI and Hoescht. We have developed a simple yet effective method to visualise mtDNA in situ within living cells using the fluorescent stain PicoGreen. Quantitative analysis shows that PicoGreen can be used to estimate the degree of mtDNA depletion within living cells. We have used this approach to study the arrangement and fluorescence of nucleoids in cells depleted of mtDNA by treatment with the anti-viral nucleoside analogue, 2',3'-dideoxycytidine. We also studied the distribution of mtDNA in fibroblasts cultured from patients with mitochondrial disease. Combining PicoGreen staining with histochemical and immunocytochemical approaches enabled us to examine the effects of mtDNA depletion on mtDNA-related components at the level of single cells. This method is able to detect an intermediate degree of mtDNA depletion in living cells, and can be used to detect mtDNA free cells (rho0 cells) in culture even at very low numbers. We have also adapted the technique to efficiently sort rho0 cells from populations of normal cells by fluorescent-assisted cell sorting (FACS), without the need for selection of respiratory competence. This should be useful for the construction of new trans-mitochondrial 'cybrid' cell lines.
Ashley
Neil
N
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. Neil.Ashley@obs-gyn.ox.ac.uk
Harris
Dot
D
Poulton
Joanna
J
D001483:Base Sequence; D002460:Cell Line; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004996:Ethidium / Q000031:analogs & derivatives*; D005347:Fibroblasts / Q000378:metabolism; D005404:Fixatives; D005456:Fluorescent Dyes; D005557:Formaldehyde; D006801:Humans; D028361:Mitochondrial Diseases / Q000378:metabolism; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction; D011108:Polymers; D013194:Staining and Labeling / Q000379:methods*; D016047:Zalcitabine / Q000494:pharmacology
D004272:DNA, Mitochondrial; D005404:Fixatives; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D011108:Polymers; C067883:dihydroethidium; D005557:Formaldehyde; D016047:Zalcitabine; D004996:Ethidium; C003043:paraform