Generating Recombinant Live-Attenuated Influenza Viruses for Vaccines
Transcript
To begin, plate a million 293T cells in each well of a 6-well plate with DMEM containing 10% fetal bovine serum and 1% pen strep. Next, prepare enough plasmid transfection mixture for 100 microliters per well. Combine a microgram of each plasmid and fill the tube with pre-warmed Opti-MEM cell medium to 50 microliters. In another tube, add twice the volume of lipofectamine 2000 corresponding to the total quantity of plasmid to be transfected, and top the tube off to 50 microliters with Opti-MEM.
Before adding the transfection mix to the cells, let it sit for 30 minutes at room temperature. Then, add 100 microliters of the transfection mix to each well and move the plate to an incubator for 16 hours. The next day, change the medium to DMEM with 0.3% BSA and 1% pen strep. Then. move the cells to 33 degrees Celsius with 5% carbon dioxide for five hours.
After five hours, add an overlay of a million influenza-permissive MDCK cells in trypsin-supplemented DMEM. Maintain the co-cultures for 2 to 3 days under the 33 degrees Celsius incubation regime to generate and amplify the virus. Later, clear out the debris using a five-minute 260 g spin, then collect the supernatants.
