Figure 2: The Cre/loxP system for generating HDAds. The HDAd contains only ~500 bp of cis-acting Ad sequences required for DNA replication (ITRs) and packaging (ψ); the remainder of the genome consists of the desired transgene and non-Ad stuffer sequences. The HDAd genome is constructed as a bacterial plasmid (pHDAd) and is liberated by restriction enzyme digestion (e.g., PmeI). To rescue the HDAd, the liberated genome is transfected into 293 cells expressing Cre and infected with a helper virus bearing a packaging signal (ψ) flanked by loxP sites. Cre-mediated excision of ψ renders the helper virus genome unpackageable, but still able to provide all of the necessary trans-acting factors for propagation of the HDAd. The titer of the HDAd is increased by serial coinfections of 293Cre cells with the HDAd and the helper virus.