A High-Efficiency Cre/loxP-Based System for Construction of Adenoviral Vectors

Adenovirus (Ad) vectors provide a highly efficient means of mammalian gene transfer and are widely used for high-level protein expression in mammalian cells, as recombinant vaccines and for gene therapy. A commonly used method for constructing Ad vectors relies on in vivo homologous recombination between two Ad DNA-containing bacterial plasmids cotransfected into 293 cells. While the utility of this two-plasmid approach is well established, its efficiency is low owing to the inefficiency of homologous recombination. To address this, we have developed an improved method for Ad vector construction based on Cre-mediated site-specific recombination between two bacterial plasmids, each bearing a loxP site. Ad vectors are generated as a result of Cre-mediated site-specific recombination between the two plasmids after their cotransfection into 293 cells expressing Cre recombinase. The frequency of Ad vector rescue by Cre-mediated site-specific recombination is significantly higher (approximately 30-fold) than by in vivo homologous recombination. The efficiency and reliability of this method should greatly simplify and expedite the construction of recombinant Ad vectors for mammalian gene transfer. Ad vectors are commonly constructed by homologous recombination between two plasmids cotransfected into 293 cells. This method has numerous advantages but results in low numbers of plaques owing to inefficient recombination. We have developed an improved method based on Cre-mediated site-specific recombination, which results in vector rescue at frequencies approximately 30-fold higher than by homologous recombination. This method should greatly simplify and expedite the construction of recombinant Ad vectors for mammalian gene transfer.