MITOCHONDRIA TRANSFER INTO FIBROBLASTS: LIPOSOME-MEDIATED TRANSFER OF LABELED MITOCHONDRIA INTO CULTURED CELLS

Mitochondria are organelles that play a key role in cellular energy metabolism through oxida-tive phosphorylation and the production of ATP. Mutations in mitochondrial DNA (mtDNA) that negatively affect the function of oxidative phosphorylation enzyme subunits or the mitochondrial protein translational mech-anisms can result in severe impairment in tissues with high energy requirements (central nervous system, heart, skeletal muscle, etc).1 Animal models of mtDNA diseases would be useful, yet techniques for efficient transfer of mitochondria are limited.2 We tested whether isolated, viable mitochondria can be efficiently transferred into cultured mouse fibroblasts using synthetic liposomes, which are used routinely to deliver molecules such as DNA and RNA into cells.3 This study targets liposome-mediated transfer of labeled mitochondria into cultured NIH/3T3 mouse fibroblasts using a mitochondrial track-ing dye. Mitochondria were isolated from the mouse liver and labeled with MitoTracker RedH CMXRos, a probe that binds selectively to the mitochondrial membrane and can be visualized using fluorescence microscopy.4 Transfer of mitochondria into cultured cells was performed using the synthetic liposome, Lipofectin (from Invitrogen). Controls consisted of MitoTracker RedH-labeled mitochondria without liposome transfection reagent, un-treated cells, and cells labeled with MitoTrack-erH Red only. Under fluorescence microscopy, transferred mitochondria were observed in fibroblasts (throughout the cytoplasm around the nucleus). Proof that viable mitochondria can be transferred into cultured cells using synthetic liposomes paves the way for adapting this technique to the transfer of genetically engineered mitochondria into embryonic stem cells for development of live heteroplasmic mice that will serve as models of human mtDNA diseases.5,