Figure 1.

Schematic outline of the AdEasy system. The gene of interest is first cloned into a shuttle vector, e.g., pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease PmeI, and subsequently cotransformed into E. coli BJ5183 cells with an adenoviral backbone plasmid, e.g., pAdEasy-1. Recombinants are selected for kanamycin resistance, and recombination was confirmed by multiple restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines, e.g., 911 or 293 cells. Recombinant adenoviruses typically are generated within 7–10 days. The “left arm” and “right arm” represent the regions mediating homologous recombination between the shuttle vector and the adenoviral backbone vector (see Materials and Methods for sequence composition). An, polyadenylation site; Bm, BamHI; RI, EcoRI; LITR, left-hand ITR and packaging signal; RITR, right-hand ITR; Sp, SpeI.