Editing 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&nbsp; 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 Ã 10^6 HEK-293 cells (at 70â80% 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 Ã g. Discard the pellet and keep the supernatant. 

13. Use all the previous centrifuged crude lysate to infect 4 Ã 10^8 HEK-293 cells (at 70â80% 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 Ã 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 â80°C.<br> 

[[Category:Protocols]]