Editing Adenovirus AdEasy (section)

== <br> Viral Production in 293 or 911 Cells  ==

<br> 1. 293 cells (E1-transformed human embryonic kidney cells) or 911 cells (E1-transformed human embryonic retinal cells) in one or two T-25 flasks at 2 x 106 cells per flask ~24 hours prior to transfection. The confluency should be about 50% to 70% at the time of transfection. 

2. On the day of transfection, digest recommbinant adenovial plasmids with PacI (usually 4 µg DNA is needed to transfect one T-25 flask). Ethanol precipitate the plasmids and resuspend in 20 µl of sterile H2O. 

3. Perform a&nbsp; transfection using GeneExpressoMax. Mix 4 µg of PacI-digested plasmid and 12 µl of GeneExpressoMax for each T-25 in 500 µl of OptiMem I or DMEM medium, and incubate at room temperature for 20 min. 

4.&nbsp; Add GeneExpressoMax-DNA mix to the flasks, and return to 37oC CO2 incubator. 

6. Remove medium containing GeneExpressoMax-DNA mix 48 hours later, and add 6 ml fresh complete DMEM (10% FBS, 1% Pen/Strep). Do not change the GeneExpressoMax/DNA medium if a significant amount of floating cells are observed; sometimes this happens with 293 cells and doesnât necessarily indicate a problem. If lots of floating cells are seen, add 6.0 ml complete DMEM to each flask and incubate at 37°C overnight. Change medium next morning. 

7. Transfections and viral productions can minotored by GFP expression if pAdTrack-based vectors are used. No obvious plaques or CPE are easily observed by standard microscopy up to 2 weeks post transfection in most cases. However, plaques are always observed under fluorescence using the GFP marker. 

8. Scrape cells off flasks with a rubber policeman (not trypsin) at 7 to 10 days post-transfection and transfer to 50 ml conical tubes. Spin cells in a benchtop centrifuge and resuspend pellet in 2.0 ml HBSS or sterile PBS. Freeze cells in dry ice/methanol bath, and thaw in a 37oC water bath. Vortex vigorously. Repeat freeze/thaw/vortex for 3 more cycles (four cycles total). Do not let virus supernatants warm up. Spin samples briefly and store supernatant at -20oC. 

9. Infect two 50% to 70% confleunt T-25 flasks of 911 or 293 cells using 30-50% of the above viral supernatants for each flask. CPE or cell lysis should become evident at 2 to 3 days post infection. Productive infections easily observed with the AdTrack vectors. 

10. Collect viruses when a third to half of the cells are detached, usually 3 to 5 days post infection. Presence of the recombinant adenoviruses can be confirmed by Western blot and/or PCR (for PCR, take 5 µl virus sup plus 10 µl PCR-grade Protease K at 55oC for 1 hour, then boil samples for 5 min. spin briefly, use 1 to 2 µl for PCR). 

11. Scrape cells off and prepare viral supernatants as described in Step 8 above. You should have at least 107 infectious particles/ml at this stage, and often much more. Each round of amplification should give at least 10-fold more virus than present in the previous round. 

12. To amplify further, repeat the infection of cells using 30-50% of the viral supernatant from Step 11, using T75 flasks instead of T25 flasks. Titers can be measured at any time, which is particularly easy with AD-Track vectors. Simply infect 293 cells with various dilutions of viral supernatant and see how many are green 18 hours later. Without AdTrack, viruses can be plaque titered or titered by limiting dilution using standard methods; we find these methods much less simple and quantitative than that employing the GFP marker, but these have to be used if GFP is not present. <br>