An enhanced system for construction of adenoviral vectors by the two-plasmid rescue method

The two-plasmid rescue method of constructing Ad vectors, which relies on e ither homologous or Cre-mediated recombination between two plasmids cotrans fected into 293 or 293Cre4 cells, respectively, offers advantages over othe r approaches because of its simplicity, We have improved the efficiency of vector construction by both homologous and Cre-mediated recombination by re placing the single ITR in the shuttle plasmid with a head-to-head ITR junct ion, We have also expanded the versatility of this method by incorporating a Cre expression cassette into the plasmids to permit high-efficiency Cre-m ediated vector rescue using 293 cells, abrogating the need for Cre-expressi ng cell lines, This new system retains the simplicity of the original but r esults in an similar to 100-fold increase in the number of recombinant viru ses produced, all of which contain the foreign DNA insert, and allows high- efficiency Cre-mediated vector isolation using any El-complementing cell li ne.