Description and production of the HD vector AdFTC/cFIX/ChMAR. (A) DNA sequences used for production of the helper-dependent (HD) vector AdFTC/cFIX/ChMAR. The canine coagulation factor IX (cFIX) expression cassette contains the human α-1-antitrypsin promoter (hAAT-p), the 2 liver specific enhancers apolipoprotein E (ApoE) and hepatocyte control region (HCR), the cFIX cDNA, and a portion of the first intron from the human F9 gene. A 16.2-kb fragment of alphoid repeat DNA fragment from human chromosome 17 is flanked by a 4.2-kb fragment containing the left terminus of adenovirus type 5 (nt 1-452), 2 copies of the immunoglobulin κ MAR (IgκMAR), and by a 7.2-kb fragment containing the HCR enhancer, a multiple cloning site (MCS), the hFIX expression cassette, and the right terminus of adenovirus type 5 (nt 35796-35935). (B) The ratio of HD vector and helper virus in final preparations was determined by Southern blot analyses. We isolated the viral DNA from the purified particles and performed a Southern blot, which was hybridized with a probe (NotI/SacI fragment from p72N5′ITRIgκMAR/SalI) that was able to visualize the ratio between helper virus genomes and HD vector genomes. Shown are 4 different preparations (lanes 3-6) of the HD vector AdFTC/cFIX/ChMAR of which only the best were used for gene transfer. As controls, the viral DNA from purified helper virus particles (lane 1) and the total DNA of C7-Cre cells at serial passage one during viral production (lane 2) were isolated. (C) The amount of transducing units in final preparations of the HD vector AdFTC/cFIX/ChMAR was determined by a quantitative Southern blot.