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Background  Construction of conditionally-replicative Adenovirus (CRAd) is complex and time-consuming. While homologous recombination (HR) using a two-plasmid system in bacteria is commonly used to generate CRAds, alternative methods may be required when HR fails. Previously,  in vitro  ligation has been suggested to facilitate construction of E1/E3-deleted, replication-incompetent Ad vectors. However,  in vitro  ligation has only rarely been used to generate CRAds and may be a complex procedure for molecular biologists who are not experts in the field.    Methods and Results  A modified  in vitro  ligation approach was developed to construct a double-targeted, E1B55k-deleted CRAd. The method allowed the incorporation of a tumor-specific promoter, e.g. the heat-shock protein 70 (hsp70) promoter, upstream of  E1a , deletion of the  E1B55k  gene, and HR-free cloning of the recombined  E1Δ55k  gene into the Ad genome. The genetic structure of the CRAd was confirmed using restriction analysis and PCR. The replication rate of the hsp70E1Δ55k CRAd was 1.5–2% of Ad without E1Δ55k deletion.    Conclusion  A 3-step cloning approach can generate a double-targeted,  E1B55k -deleted CRAd using a straight-forward, modified  in vitro  ligation procedure.

A simplified in vitro ligation approach to clone an E1B55k-deleted double-targeted conditionally-replicative adenovirus