Generation and Amplification of Recombinant Adenovirus
Recombinant adenovirus vectors are replication-defective and, thus, they must be produced and propagated in cell lines that complement the defect. Still, these vectors must be handled following Biosafety Level 2 practices, just like the wild-type adenoviruses from which they are derived. The reason is that replication competent adenoviruses (RCA) may result from rare recombination events between the recombinant vector genome and the adenoviral sequences present in HEK293 cells during the amplification process.
Conventional approaches to producing small volumes of viral vectors involve culturing cells in stationary and adherent cultures. The main protocols of this small-scale production method are presented below. These recombinant adenoviruses can be used in experimental procedures involving cells or laboratory animals, as well as a starting material for subsequent rounds of larger amplification.
- Twenty-four hours before transfection, plate 106 HEK-293 cells/well in a 6-well cell culture plate with DMEM + 10 % FBS (Fetal Bovine Serum). Three wells are needed for each adenovirus vector, one of them as control.
- Digest 100 μg recombinant adenoviral plasmid with 15 U Pac I for 5 h to separate the adenovirus genome from the remaining plasmid sequences.
- Precipitate digested DNA with 0.1 volume of sodium acetate and 2.5 volumes of ethanol and resuspend in 100 μL sterile TE buffer.
- Perform a standard transfection using 6 μg Pac I digested plasmid per 106 HEK293 cells. Incubate at 37 °C and 5% CO2 for 3 days.
- Harvest medium and cells. Freeze at −80 °C and thaw at 37 °C three times to lyse the cells and release the adenoviruses.
- Centrifuge at room temperature for 5 min and 1,200 g and keep the supernatant. Discard the pellet.
- Use all the obtained supernatant to infect 7 × 106 HEK293 cells (at 70–80 % confluency) in a 10-cm plate, in a final volume of 8 mL, with DMEM + 2% FBS.
- Incubate at 37 °C and 5 % CO2 until general cytopathic effect is observed (usually 4-9 days).
- Harvest medium and cells. Freeze-thaw three times to release adenovirus from cells. Centrifuge at room temperature for 5 min and 1,200 g. Discard the pellet and keep the supernatant.
- Use 1 mL of the supernatant obtained to infect a total 3.5 × 108 HEK293 cells in twenty 15-cm plates (70-80% confluency), in a final volume of 14 mL/plate with DMEM + 2% FBS.
- Incubate at 37 °C and 5% CO2 until general cytopathic effect is observed.
- Harvest medium and cells. Distribute it among six 50-mL Falcon tubes. Centrifuge at room temperature for 5 min at 620 g. Keep 19 mL supernatant and discard the rest.
- Resuspend the pellets with the reserved 19 mL supernatant. Freeze-thaw three times to release adenovirus from cells.
- Centrifuge at room temperature for 5 min at 1,200 g. Discard the pellet and keep the supernatant. The supernatant can be tested for Mycoplasma contamination as a quality control. Store at −80 °C.