Rapid Isolation of Mitochondrial DNA-Depleted Mammalian Cells by Ethidium Bromide and Dideoxycytidine Treatments

Abstract

Mitochondrial DNA (mtDNA)-depleted (ρ 0) cells are often used as mtDNA recipients to study the interaction between the nucleus and mitochondria in mammalian cells. Therefore, it is crucial to obtain mtDNA-depleted cells with many different nuclear backgrounds for the study. Here, we demonstrate a rapid and reliable method to isolate mammalian mtDNA-depleted cells involving treatment with the antimitochondrial agents ethidium bromide (EtBr) and 2',3'-dideoxycytidine (ddC). After a short exposure to EtBr or ddC, followed by rapid clonal isolation, we were able to generate viable mtDNA-depleted cells from mouse and human cells and were able to successfully repopulate them with exogenous mitochondria from platelets isolated from mouse and human blood samples. These mtDNA-depleted cells can be used to characterize the nuclear mitochondrial interactions and to study mtDNA-associated defects in mammalian cells. Our method of isolating mtDNA-depleted cells is practical and applicable to a variety of cell types.

Effect of mtDNA depletion from C6 glioma cells and characteristics of the generated C6ρ0 cells

“…The mitochondrial genome is essential for a normal cellular function and it encodes important subunits of the mitochondrial respiratory chain and F0F1-ATPase, which are required for oxidative phosphorylation. Depletion of mtDNA results in the inhibition of the mitochondrial respiratory chain and the loss of mtDNA-encoded mitochondrial enzyme activity (16,(22)(23)(24).…”

“…Single cell clones were isolated and collected by the limiting dilution method ( 16 ) when the mtDNA band disappeared. When no mtDNA fragment was detected by PCR, the treated cells were quickly plated into 96-well plates at a density of one cell per well to isolate pure cell clones derived from the heterogeneous colony.…”

“…Fortunately, hybrid technology provides a new strategy to study diseases associated with mitochondrial dysfunctions. The technique is based on the use of a cell line in which the mtDNA is depleted (ρ0 cells) and can be used as a mitochondrial receptor when fused with platelets ( 15 , 16 ). Since cytoplasmic hybrids (cybrids) have the same genetic background, biochemical analysis is made possible.…”