To rescue viruses the constructed plasmids were digested with PacI and two mg DNA were transfected (Lipofectamine 2000 Reagent, Invitrogen, Carlsbad, CA) into the Ad-E1-expressing HEK293 cells. Following plaque formation, they were processed for large-scale propagation in HEK293 cells. Viruses were purified by double cesium chloride ultracentrifugation and dialyzed against phosphate-buffered saline containing 10% glycerol. Viruses were stored at 280uC until use. Final aliquots of virus were analyzed for physical titer using absorbance at 260 nm. The infectious viral titer (IFU) per ml was determined by tissue culture infectious dose (TCID 50 ) assay. The TCID 50 titer was calculated by using KARBER statistical method: T TCID 50 titer = 10610 1 + d(S 2 0.5) /ml, in which d is the log 10 of the dilution and S is the sum of ratios from the first dilution. Modifications of the hexon gene was confirmed by PCR analysis with the primers 59HVR2 (sense), CTCACGTATTTGGG-CAGGCGCC and 3'HVR5 (antisense), GGCATGTAAGAAA-TATG AGTGTCTGGG, which anneal up and downstream of the site of the insertion within the hexon open reading frame.