293A Cell Line

Most of the adenovirus is contained within the floating cells and is not released into the medium until those cells burst. We recommend changing the medium every 3 days or so until it is obvious that a lot of cells become big and rounded and are detaching from the plastic. Once a cell bursts, the free viruses rapidly infect the neighboring cells. If you're ever worried that you're losing infected cells (and therefore potential virus) in your medium changes, you can always save the medium with the floating cells, freeze/thaw it 3 times and then use a little (maybe 1/10th) and add it back to your culture with fresh media. Or, replace only half of the medium with fresh medium and do this more often than every three days.

Any 293-derived cell line or other cell line that expresses the E1 proteins may be used to produce adenovirus. In 293A cells (recommended for adenovirus production), "A" stands for "adherent" because the 293A cells (which are just a single-cell clone of regular 293) tend to adhere and form nice flat monolayers in tissue culture dishes. This is why they work so well for plaque assays. Regular 293 cells will not form the same type of monolayers; they exhibit holes and gaps during growth.

We use mycoplasma-tested Gibco FBS (Cat. No. 16000-044) and use the following plasticware for 293A cells:

T175: Fisher Cat. No. 10-126-13; this is a Falcon flask with a 0.2 µm vented plug seal cap.

T75: Fisher Cat. No. 07-200-68; this is a Costar flask with a 0.2 µm vented seal cap.

100 mm plate: Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate.

We get excellent adherence on these plates under routine cell culture/maintenance conditions (expect cell lysis in 293A cells when making adenovirus).

Before you can transfect your expression clone into 293A cells, you must expose the left and right viral inverted terminal repeats (ITRs) on the vector to allow proper viral replication and packaging. This also removes bacterial sequences (i.e., pUC origin and ampicillin resistance gene). Both pAd/CMV/V5-DEST and pAd/PL-DEST ;vectors contain Pac I restriction sites (see maps on pages 20 and 22 of the manual (http://tools.thermofisher.com/content/sfs/manuals/pad_dest_man.pdf), respectively, for the location of the Pac I sites).

Note: Make sure that your DNA sequence of interest does not contain any Pac I restriction sites. If you are unable to use the Pac I site, you can use the Swa I site.

No. The ViraPower system uses adenovirus type 5. Adenoviruses (Adenoviridae) and adeno-associated viruses (Parvoviridae) are completely different. Adeno-associated viruses are often associated with adenovirus infections, hence the name. Since they are thought to be virtually non-pathogenic, they are attractive vectors for gene therapy. The disadvantage is that they can package only about half the foreign DNA that adenoviruses can.