Construction of First Generation Adenovirus Vectors
First generation (FG) vectors typically have deletions in two regions of the viral genome: Early region 3 (E3) which is nonessential for virus replication in cultured cells, and Early Region 1 (E1) which is essential but whose functions are provided in trans by 293 cells. Packaging capacity of foreign DNA inserts in such vectors is approximately 7-8 kb. There are numerous ways to construct FG vectors but the simplest, and most efficient is based on cotransfection of two plasmids, one which is relatively small, called a shuttle plasmid, carrying the cassette for expression of a foreign gene, and the other a large genomic plasmid consisting of most of the Ad genome. Neither plasmid is infectious and virus is only produced by recombination between the two plasmids. The most efficient method for achieving this recombination event is one catalyzed by a site-specific recombinase such as Cre recombinase. AdVec has developed and made available one of the most used such systems called AdMaxTM in which the genomic plasmid encodes the Cre recombinase (or the FLP recombinase from yeast) and the shuttle plasmid carries the expression cassette. The system is illustrated below. More detailed information and protocols are available from DMKS Research (email: DMKS_Ltd@shaw.ca).