Figure 3

Cloning of oncolytic adenoviruses Ad.Ki•COX and Ad.COX•Ki. Panel A: The structure of the recombined adenoviral plasmid pAd.Ki•COX is shown. Restriction endonucleases used for analysis are mapped. For clarity, a map of pAd.COX•Ki, which is with the exception of the swapped promoters identical to pAd.Ki•COX, has been omitted. Panel B: BJ5183 cells pre-transformed with pAdEasy-1 were transformed with pShuttle#4 Ki•COX and pShuttle#4 COX•Ki. The integrity of the pAdEasy-1 plasmid in the pre-transformed E. coli was confirmed by Hind III digestion (data not shown). For each construct ten small scale DNA plasmid preparations of kanamycin resistant clones were digested with Hind III and Pac I and analyzed by electrophoresis through a 0.8% agarose gel and ethidium bromide staining. Panel C: High quality DNA was prepared by CsCl banding of potential valid recombinants and digested with Pac I, Spe I/Swa I, Cla I, and Hind III. The heterologous promoter driving the viral E4 region was flanked by Spe I and Swa I and the promoter controlling E1 by Cla I. The adenoviral backbone plasmid pAdEasy-1 served as a control. Diagnostic fragments obtained with each enzyme are marked with an asterisk (*). Panel D: Standard PCR was used to confirm on the rescued infectious recombinant adenoviruses Ad.Ki•COX and Ad.COX•Ki the presence of the Ki-67 and COX-2 promoter with the upstream polyA sequences in each virus. Ad5 served as negative control. The 1 kb DNA ladder from Invitrogen/Gibco was used as DNA size marker (M).