PAd5-Blue: Direct ligation system for engineering recombinant adenovirus constructs

Abstract

We have engineered a new vector that makes use of direct ligation for the generation of replication-defective recombinant adenovirus constructs. In the pAd5-Blue vector, unique yet common restriction endonuclease sites exist, that allow cloning in a directional manner of a gene of interest under control of a cytomegalovirus promoter, upstream of a simian virus 40 polyadenylation signal. The insertion of the new gene replaces the β-galactosidase α gene fragment in the pAd5-Blue vector, allowing the identification of recombinants in bacterial culture by the selection of white colonies. Plasmid DNA from white colonies is digested with PacI and transfected into 293 cells, resulting in the generation of a homogenous population of adenovirus containing the gene of interest. The pAd5-Blue vector system does not rely on recombination either in mammalian or bacterial cells. Furthermore, because of compatible overhangs, the variety of restriction endonucleases that can be used to generate the inserted gene gives flexibility to the process for greater ease of use. The system is quick and straightforward, allowing the generation of recombinant adenoviruses within three weeks of obtaining an appropriate insert. This new vector should greatly enhance the ease and speed with which new recombinant adenovirus constructs can be made.