Construction of Recombinant Adenovirus Genomes by Direct Cloning

This protocol describes how to generate an infectious adenovirus vector by direct ligation and cloning. This is the first step in the production of a recombinant adenovirus vector. The protocol begins with a convenient and efficient double-selection procedure based on antibiotic resistance and identification of green fluorescent protein (GFP)-negative bacterial colonies. In this protocol, the prokaryotic expression cassette for GFP in the shuttle plasmid pSh-pkGFP is replaced with the transgene. The resulting pShuttle-transgene plasmid is then used to clone the transgene expression cassette into pAd-pkGFP, using the same double-selection procedure.