I have made adenoviruses with fluorescent proteins using the Invitrogen ViraPower Adenoviral Expression System. Both of them show excellent expression when transfecting HEK cells with the destination vector, but when I try to use the adenovirus I see very poor gene expression. I believe this points to an issue with my harvesting procedure. After scraping the cells I put the suspended cells and medium into a 50 mL tube and put them through 3 freeze thaw cycles with 30 minutes of freezing and 15 minutes of thawing @ 37C. After this I centrifuge the cells for 15 minutes at 3000g and filter sterilize the supernatant with a 0.22um filter. I have not titered the adenoviruses, but I used as much as 100uL in 2mL of medium to transduce HeLa cells (~50% confluence) in a 24 well plate and saw very little expression via confocal.