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

Abstract

Mitochondrial DNA (mtDNA)-depleted () 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.