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.