Cyber generation is still the state of the art protocol to understand the pathogenic role of any novel mitochondrial DNA mutation and to correlate the percentage of atriplasmy with the severity of the disease. This technique allows performing biochemical investigation in a homogeneous nuclear system, in which the contribution of the nuclear background of the patient is absent. This technique allows validating the pathogenicity of a mutation of the mitochondrial DNA.
and makes it possible to study its impact at biochemical level Wash the 35 millimeter dishes containing the fibroblasts twice using two milliliters of 1X PBS without calcium and magnesium. Clean the outer surface of the dishes with 70%ethanol, and wait until the alcohol evaporates. Remove the lids from the dishes and the screw caps from the bottles.
Place each dish without the lid upside down at the bottom of each 250 milliliter centrifuge bottle. Slowly add 32 milliliters of a nucleation medium to each bottle allowing the medium to enter the dish and come in contact with the cells. Remove any bubbles from the dishes using a long glass pasture pipette, curving the tip in a bunsen flame.
Close each bottle with the screw cap, and transfer them to the centrifuge. Centrifuge for 20 minutes at 37 degrees Celsius and 8, 000 x G.Aspirate the medium from the bottles and discard it. Remove the dishes by inverting the bottles on a sterile gauze previously sprayed with 70%ethanol.
Clean the outer surface of the dishes and their lids with 70%ethanol, and close the dishes after the ethanol evaporates. Before proceeding check for cytoplast formation using an inverted microscope for live cells. Look for extremely elongated fibroblasts due to the extrusion of their nuclei induced by cytochalasin.
To each 35 millimeter dish add 1, 000, 143 row zero cells. Re-suspended in two milliliters of culture medium supplemented with 5%FBS. Leave the dishes for three hours in a humidified carbon dioxide incubator to settle the 143 row zero cells on the ghosts.
After three hours of incubation, aspirate and discard the medium. Wash the adherence cells twice with two milliliters of DMEM high glucose medium without serum or MEM, then aspirate and discard the medium. Add 500 microliters of polyethylene glycol solution to the cells and incubate for exactly one minute.
Aspirate and discard the polyethylene glycol solution. Wash the cells three times with two milliliters of DMEM high glucose without serum or with MEM. Add two milliliters of fusion medium, and incubate overnight in the incubator at 37 degrees Celsius with 5%carbon dioxide.
Trypsinize the cells in the remaining culture dishes Count them and seed them into one or more Petri dishes Add 50 to 100 cells per dish in the supplemented culture until clones appear. Then let them grow for several days. Using a stereo microscope, pick up the clones from the Petri dish with cloning cylinders or a pipette tip.
Try to avoid pooling different clones and transfer them to a 96 well plate, each well containing 200 microliters of supplemented culture medium. Cybrids were generated starting from fibroblasts derived from a patient carrying the heteroplasmic M2343A2G One of the most common mitochondrial DNA mutations associated with mitochondrial myopathy, encephalopathy lactic acidosis, and stroke-like episodes. Analysis of a variable number of tandem repeats showed that cyber DNA is identical to the row zero cells confirming the replacement of the patient's cyber DNA with the 143 B genome.
Restriction fragment length polymorphism, or sequencing analyses, can be used to assess the presence of the mitochondrial DNA mutation and the heteroplasmy percentage. When attempting this protocol it is important to verify the correct genetic asset of the nucleon and mitochondrial DNA, as well as the mitochondrial DNA amount in the repopulated cybrids.
