Figure 1

Overall Schematic diagram of the 3-step cloning strategy to generate CRAd-hsp70E1Δ55k using a modified in vitro ligation system. To generate a double-targeted CRAd, a TSP-regulated E1ΔE1B55k fragment was reintroduced into an E1/E3 deleted Ad backbone. a, the 383-bp human hsp70 promoter was cut with SalI, Klenow-dNTP blunted, XhoI digested, gel purified and inserted into the EcoRV/XhoI site in pMKE1 (b), to replace the MK promoter and generate phsp70E1 (c). Next, to remove the E1B55k fragment, phsp70E1 was digested with XhoI, Klenow dNTP blunted and digested with KpnI (c). Simultaneously, to remove the CMV promoter yet maintain the polyA signal, pShuttle from the Adeno-X system was digested with MfeI, Klenow dNTP blunted and digested with KpnI (d). Thus, the pShuttle of the AdenoX system was modified to encode a CMV-promoter deleted, TSP-regulated, E1B55k-deleted E1 gene in phsp70E1Δ55k (e). Next, to generate the recombinant CRAd genome, pAdhsp70E1Δ55k, the hsp70E1Δ55k fragment was removed from phsp70E1 with I-CeuI and PI-Sce-I digestion, purified via agarose gel electrophoresis and ligated into the pre-I-CeuI/PI-Sce-I-digested Ad backbone plasmid (f). SwaI digestion eliminated the chance of religation of the Ad backbone plasmid without recombination. MK promoter, the human 2.6 kb gene promoter. CMV promoter, the human cytomegalovirus immediate-early gene promoter. BGH polyA, the bovine growth hormone early mRNA poly-adenylation signal. Grey-filled arrows indicate fragments excised for further cloning.