New tools for the generation of E1-and/or E3-substituted adenoviral vectors

We have designed new vectors for the construction of recombinant adenovirus es containing expression cassettes in the El and/or E3 regions. Using a ver satile set of restriction enzymes, the cassettes are cloned into small bact erial vectors and subsequently introduced into large plasmids containing th e adenoviral sequences. Two positive selection markers facilitate the recov ery of a cosmid containing a copy of the sequence of the recombinant adenov irus. The resulting cosmid is transfected into 293 or 911 cells in order to rescue the virus. Importantly, the method does not require any recombinati on event, either in E. coil or in mammalian cells. The entire procedure can generate viral plaques in 12 days.