Adenovirus Prestock

1. 6 to 16 hours before transfection, plate 10^6 293A cells in several 25-cm2 tissue culture flask(s) with DMEM + 10% FBS. Plate at least two flasks. One plate may work as control plate and help to follow vector amplification.

2. Digest 1 ug of recombinant pAd5B (backbone) adenoviral plasmid and pAd5 plasmid (with GOI) with 30 U of PacI to separate adenovirus genome from bacterial sequences.

To ensure complete digestion, 6 h later add another 30 U of PacI. Digest overnight.

3. Precipitate digested DNA with two volumes of ethanol and resuspend in 20 mL of sterile ddH2O.

4. Perform a standard transfection using 2 ug of PacI digested plasmids  per 10^6 HEK-293 cells.

5. Incubate the DNA with GeneExpressoMax transfection reagent for 20 min at room temperature.

6. Add DNA complexes dropwise to the cells. Incubate at 37°C and 5% CO2 for 6 days.

7. Scrape/harvest the medium and cells. Freeze/thaw three times to release adenovirus from cells.

8. Centrifuge at 4 °C for 10 min and 500 x g and discard the pellet.

9. Use all the centrifuged crude lysate to infect 8 × 10^6 HEK-293 cells (at 70–80% confluency) in a 10-cm plate.

10. Incubate at 37°C and 5% CO2 until general cytopathic effect is observed (usually between 3 and 6 days).

11. Harvest medium and cells. Freeze/thaw three times to release adenovirus from cells.

12. Centrifuge at 4°C for 10 min and 500 × g. Discard the pellet and keep the supernatant.

13. Use all the previous centrifuged crude lysate to infect 4 × 10^8 HEK-293 cells (at 70–80% confluency) in twenty 25-cm plate.

14. Incubate at 37°C and 5% CO2 until general cytopathic effect is observed (usually between 32 and 38 h).

15. Harvest medium and cells. Freeze/thaw three times to release adenovirus from the cells.

16. Centrifuge at 4°C for 10 min and 500 × g. Discard the pellet and keep the supernatant (viral-prestock).

17. Titer the viral pre-stock by anti-hexon staining method.

18. Aliquot and store viral pre-stock at −80°C.