A simple method for the rapid generation of recombinant adenovirus vectors.
Recombinant adenoviruses are useful vectors for basic research. When the vectors are used for delineating protein function, several viruses, each containing a mutated version of the transgene are compared at the same time. However, methods to generate multiple vectors simultaneously within a short time period are cumbersome. In this report, we show that a novel backbone plasmid, when cotransfected with routinely used shuttle vectors into HEK293 cells allowed for production of recombinant viruses in an average of 14 days. The recombinant viruses had no detectable wild-type virus contamination by A549 plaque assay and only three to 300 E1a copies per 109 adenovirus genomes by a sensitive PCR-based assay. Further culturing or serial amplification did not result in wild-type revertants nor did cultures show increased levels of E1a copy number by quantitative PCR. Thus, recombinant adenovirus vectors can be produced very simply, rapidly and with little to no contaminating wild-type particles. This system should facilitate the generation of multiple genetic variants by eliminating the need for time-consuming plaque purification and the need to manipulate and screen very large plasmids. We call this the RAPAd.I system.
Anderson
R D
RD
Department of Internal Medicine, University of Iowa, Iowa City 52242, USA.
Haskell
R E
RE
Xia
H
H
Roessler
B J
BJ
Davidson
B L
BL
D000256:Adenoviridae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans; D010957:Plasmids; D014162:Transfection