EFFICIENT GENERATION OF RECOMBINANT ADENOVIRUS VECTORS BY HOMOLOGOUS RECOMBINATION IN ESCHERICHIA-COLI

Despite recent technical improvements, the construction of recombinant adenovirus vectors remains a time-consuming procedure which requires extensive manipulations of the viral genome in both Escherichia coli a nd eukaryotic cells, This report describes a novel system based on the cloning and manipulation of the full-length adenovirus genome as a st able plasmid in E, coli, by using the bacterial homologous recombinati on machinery. The efficiency and flexibility of the method are illustr ated by the cloning of the wild-type adenovirus type 5 genome, the ins ertion of a constitutive promoter upstream from the E3 region, the rep lacement of the El region by an exogenous expression cassette, and the deletion of the El region. All recombinant viral DNAs were shown to b e fully infectious in permissive cells, and the modified E3 region or the inserted foreign gene was correctly expressed in the infected cell s.