Editing Adenovirus pAd52 (section)

== High Titer Adenovirus Production<br> ==

General comments. Many experimental studies can be carried out using virus in the form of crude infected cell lysates. The following protocol results in high titre virus preparations. Recombinant Ads can be produced using either monolayer (see above) or suspension cultures. However, due to the greater ease of handling suspension cultures, these are preferable for the production of large amounts of high-titer viral stocks. Suspension adapted 293 cells (293N3S) are available some vendors<br>

Protocol<br>1. For infection with replication-defective viruses (E1 insertion recombinants) grow 293N3S cells in spinner culture to a density of 2-4 x 105 cells/ml in 4 liters complete Joklik’s modified MEM + 10% HS. For growth of wt adenovirus or replication-proficient E1+ viruses (eg E3 insertion recombinants), grow 4 liters KB cells to a density of 5-6 x 105 cells/ml in complete Joklik’s modified MEM + 10% NBS. Centrifuge cell suspension at 750 g for 20 min, saving half of the conditioned medium. Resuspend the cell pellet in 0.1 vol fresh medium, and transfer to a sterile 500-ml bottle containing a sterile stir bar.

2. Add virus at MOI of 10-20 PFU/cell and stir gently at 37oC. After 1 hr, return the cells to the 4-liter spinner flask and bring to the original volume using 50% conditioned medium and 50% fresh medium. Continue stirring at 37oC.

3. Monitor infection twice daily by inclusion body staining as follows:

a. Remove a 5-ml aliquot from the infected spinner culture. Spin for 10 min at 750 g and resuspend the cell pellet in 0.5 ml of 1% sodium citrate.<br>b. Incubate at room temperature for 10 min; then add 0.5 ml Carnoy’s fixative and fix for 10 min at room temperature.<br>c. Add 2 ml Carnoy’s fixative, spin 10 min at 750 g, aspirate, and resuspend the pellet in a few drops of Carnoy’s fixative. Add one drop of fixed cells to a slide and air-dry for about 10 min; then add one drop orcein solution and a coverslip and examine in the microscope. Inclusion bodies appear as densely staining nuclear structures resulting from accumulation of large amounts of virus and viral products at late times in infection. A negative control should be included in initial tests.

4. When inclusion bodies are visible in 80-90% of the cells (2 to 3 days depending on the input MOI), harvest by centrifugation at 2000 g for 20 min in sterile 1-liter bottles. Combine pellets in a small volume of medium, and spin again. Resuspend pellet in 20 ml PBS++ and 10% glycerol. Freeze and thaw 2-3 times then aliquot and store at -70oC until use. Titrate by plaque assays on 293 monolayer cells.

Solutions

1. Complete Joklik’s modified MEM + 10% HS (or 10% NBS): Add 50 ml HS (or NBS) to 450 ml complete Joklik’s modified MEM (described in Section B). Store at 4oC.

2. 1% sodium citrate: Dissolve 1 g trisodium citrate dihydrate in H2O to a final volume of 100 ml.

3. Carnoy’s fixative: Add 25 ml glacial acetic acid to 75 ml methanol.

4. Orcein solution: Add 1 g orcein dye to 25 ml glacial acetic acid plus 25 ml H2O. Filter through Whatman No. 1 paper.<br>

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