Chapter 4 Adenoviral Vector Construction I Mammalian Systems

This chapter describes the construction of adenoviral vectors for mammalian systems. Ads are converted into mammalian gene transfer vectors by replacing the El region with the foreign DNA of interest. All methods for manipulating Ad genomes for construction of vectors rely on the observation that purified viral DNA is infectious. Early methods for generating recombinant Ad involved direct manipulation of viral DNA extracted from virions. However, a major limitation of these methods was that precisely defined alterations could not be introduced into the genome owing to the difficulty inherent in manipulating the large linear viral DNA. The two-plasmid rescue system method was developed to overcome the limitations. In this method, recombinant Ad vectors are generated by in vivo homologous recombination between two noninfectious plasmids cotransfected into 293 cells. Since its development, the two-plasmid rescue method has been widely used due to its simplicity.