Applied Viromics uses its advanced platform technology to produce adenovirus. We have optimized the viral production system to completely avoid using helper virus, thus eliminating any chance of helper virus contamination.
1) Sub-clone your gene of interest (GOI) into Applied Viromics’pAd-shuttle vector.
After subcloning your GOI, we amplify the plasmid ready for transfection.
2) Generate Adeno virus seed by introducing the shuttle plasmidinto producing cells together with corresponding helperplasmid.
293 cells are co-transfected with the shuttle plasmid containing your GOI and the pAd-helper plasmids. The P0 Adenovirus will be harvested in about 2 weeks.
3) Amplify the Adenovirus
Re-infect the 293 cells with P0 generation of Adenovirus to get the P1 generation. Perform the same action for P1 to get the P2 generation. Harvest the P2 generation of Adenovirus and proceed to purification.
4) Purification of Adenovius
We use double CsCl gradient to purify the Adenoviurs. Then de-salting step was applied to exchange the Adenovirus into the storage buffer.
Viral capsid can only pack its genome up to certain size. For recombinant viral vectors, some viral sequences are removed to accommodate the expression cassette for the gene of interest. Thus the size of the gene, together with its promoter and poly-A sequence, cannot exceed certain limit.
The maximum size of the gene of interest to be cloned into the Applied Viromics pAd-shuttle vector is 5.3kb.
Adeno Virus will lose half of its activity when stored at4°C for 7 days. We recommend aliquoting the virus into small volumes (preferably single-use aliquots) upon receipt and storing at -80°C.
According to the guidelines from the National Institutes of Health (NIH), Adeno vectors are considered as risk group 1 agents that are not known to be associated with disease in healthy adult humans. Adeno vectors can be handled in a biosafety level 1 (BSL-1) environment.