Chapter 53 Construction and Propagation of Human Adenovirus Vectors

This chapter describes methods for inserting foreign genes into the adenoviruses (Ad) genome and for purifying, growing, and titrating the recombinant viruses. Adenoviruses (Ads), which have been used extensively as a model system for molecular studies of mammalian cell DNA replication, transcription, and RNA processing, are now being increasingly investigated as potential mammalian expression vectors for gene therapy and for recombinant vaccines. In a wild-type infection, early genes are expressed prior to DNA replication, and the late gene expression, driven predominantly by the major late promoter at 16 map units, occurs after the initiation of DNA replication. Cremediates recombination between an IoxP site downstream of the E1 insertion site in the shuttle plasmid and an IoxP site in the E1 region of the genomic plasmid. E1 replacement vectors, by far the most common, are constructed by insertion of a transgene expression cassette into the E1 shuttle plasmid and subsequent rescue by recombination with the genomic plasmid in 293 cells.