A simple method for the rapid generation of recombinant adenovirus vectors

Recombinant adenoviruses are useful vectors for basic research. When the ve ctors are used for delineating protein function, several viruses, each cont aining a mutated version of the transgene are compared at the same time. Ho wever, methods to generate multiple vectors simultaneously within a short t ime period are cumbersome. In this report, we show that a novel backbone pl asmid, when cotransfected with routinely used shuttle vectors into HEK293 c ells allowed for production of recombinant viruses in an average of 14 days . The recombinant Viruses had no detectable wild-type virus contamination b y A549 plaque assay and only three to 300 Ela copies per 10(9) adenovirus g enomes by a sensitive PCR-based assay. Further culturing or serial amplific ation did not result in wild-type revertants nor did cultures show increase d levels of Ela copy number by quantitative PCR. Thus, recombinant adenovir us vectors can be produced very simply, rapidly and with little to no conta minating wild-type particles. This system should facilitate the generation of multiple genetic variants by eliminating the need for time-consuming pla que purification and the need to manipulate and screen very large plasmids.