I am trying to produce AAV-2 using Cell Biolabs kit in four 15 cm plates of HEK-293 using lipofectamine. I am using plasmid ratio of 2:1:1 (Helper : GFP : RepCap) with 12, 6 and 10 ug each plasmid. Then purification by affinity based kit method. But I am unable to detect viral protein by SDS/PAGE or viral DNA by QuickTitre kit. Please guide.
AAV is more difficult to produce. It is difficult to determine which variable in such cases, since there are so many. But I think you should try and do this with a helper VIRUS first, rather than just RepCAP. If you can get AAV with helper virus, then you have a working system. You can then blame the RepCAP. The RepCap is good to make sure you have no contamination from helper virus, but helper virus can be used for trouble shooting.
Second, dont use GFP as your GOI. GFP has variable effects in viral replication, and its best to use even an empty plasmid until it is working, if you are going to use SDS-PAGE or look for antigen via ELISA.
And AAV replicates slowly. So give it more days than usual (within limits of cell growth).
Good luck !
Instead of AAV2 try the AAV9. AAV9 doesn't have side effect. I think AAV2 isn't that much success in clinical trial.
Reference Standard provided by ATCC gave us a positive control for Western Blot Analysis. Over few days, the reactivity of positive control (from different aliquots) faded. Is there any stability related issues with AAV capsids.
Thanks for the suggestioins, I have followed the protocols from literature and used linear PEI. Now I am getting a good yield of 10^11 per ul but it seems the filled capsids are only 3%.
Any comments on enhancing the encapsidation of viral genome.
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