Adenoviral vectors are a versatile research tool with a number of positive aspects. The recombinant viruses can infect dividing and non-dividing cells, support transient, high-level protein expression, and are also easily amplified and purified to high concentrations (1x1010pfu/mL-1x1011pfu/mL). They can accommodate inserts of up to 8 kb under normal circumstances. Larger inserts are possible, provided that an equivalent part of the viral genome has been properly deleted. The Viral Vector Core (VVC) is equipped to produce adenoviral vectors with inserts up to 10kb.
The University of Iowa VVC developed and currently uses the RAPAdTM system (U.S. Patent No. 6,830,920 B2). A description of the system can be found in "A simple method for the rapid generation of recombinant adenovirus vectors" published in Gene Therapy 7:1034-1038, 2000. This system eliminates many of the challenges of standard adenoviral production. The RAPAdTM construction plasmids have left-hand ITR, E1a, and partial E1b sequence deletions which greatly reduces wild type occurrence in the final vector preparations. There is, therefore, no need for time-consuming plaque purification steps. All virus preps are tested for replication competent adenovirus (RCA) contamination by immuno-staining procedures.
The RAPAdTM system uses a two plasmid transfection system for initial viral rescue, the viral shuttle plasmid and the viral backbone plasmid. The viral shuttle plasmid contains the gene of interest that the recombinant virus will produce, as well as a few viral protein sequences. The viral backbone contains the remaining viral proteins as well as a region of homology to the viral shuttle plasmid. After linearization and transfection into HEK293 cells (which provide the needed E1 and E3 proteins), the plasmids undergo homologous recombination to produce a full length adenoviral genome with the gene of interest inserted into the E1 region. The VVC has a number of backbones with additional E3 deletions that have reporter genes (i.e. eGFP, Beta-gal, mCherry, etc) inserted into them or that can be used for inserts larger than 8kb. Using viral backbones with reporters already inserted into the E3 region allows for reporter gene function without any additional cloning. This system also offers the possibility of expressing two separate proteins with custom made backbones. For more information contact firstname.lastname@example.org.