For transmitochondrial cybrid generation, use the mitochondrial recipient cells that have been depleted to mitochondria by Rhodamine 6G treatment, and perform fusion with mitochondria isolated from the mitochondria donor cells. Ensure proper mitochondrial function abolishment in recipient cells and organelle purification from donor cells by seeding a small number of Rhodamine 6G-treated cells and isolated mitochondria in complete culture medium in a six-well plate. Check for no surviving cells remaining in the wells after one month of culture.
To proceed with the fusion, carefully add the Rhodamine 6G-treated cells to the isolated mitochondria pellet. Then centrifuge at 520g for five minutes to allow the cells to mix with the mitochondria. Add 100 microliters of 50%polyethylene glycol and gently resuspend the pellet for 30 seconds Then allow the suspension to rest untouched for another 30 seconds.
Finally, transfer the mix into a six-well plate with fresh complete cell culture medium, and place it in the incubator at 37 degrees Celsius with 5%carbon dioxide. Usually, after about one week, transmitochondrial cybrids should start growing, giving rise to clones that can be individually selected or mixed in a pool prior to their analysis. The restriction fragments obtained from the restriction fragment length polymorphism, or RFLP, analysis of wild-type mitochondria differed from the mutant one.
The RFLP analysis of the new transmitochondrial cell lines indicated that restriction fragments were identical to those obtained in their respective mitochondrial donors and different from those generated with the recipient cell lines. Further, the difference in a typical DNA nuclear genotyping profile of the two cell lines used in the hybridization process could also be used to confirm nuclear DNA purity.
