| Summary: | <p>Abstract</p> <p>Background</p> <p>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, <it>in vitro </it>ligation has been suggested to facilitate construction of E1/E3-deleted, replication-incompetent Ad vectors. However, <it>in vitro </it>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.</p> <p>Methods and Results</p> <p>A modified <it>in vitro </it>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 <it>E1a</it>, deletion of the <it>E1B55k </it>gene, and HR-free cloning of the recombined <it>E1Δ55k </it>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.</p> <p>Conclusion</p> <p>A 3-step cloning approach can generate a double-targeted, <it>E1B55k</it>-deleted CRAd using a straight-forward, modified <it>in vitro </it>ligation procedure.</p>
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