I have produced recombinant AAV virus using Agilent's AAV Helper-free kit (transfected 293 cells with pHelper, pRep/Cap, and plasmid with LTR-flanked gene of interest, freeze/thaw cells 72hr post-transfection, collect cell lysate).
Now, I would like to do an intra-vitreal injection in mice to infect the retinal ganglion cells. Is purification/concentration always required before in vivo use? If so, I'd like to know what the easiest/cheapest/most simple method is currently. I have read that you can do CsCl or iodixanol gradients (Zolotukhin et al, 1999). Is one easier or simpler than the other? Do both need to be followed by some type of purification and/or concentration step? It seems like 1999 was a long time ago and there must have been improvements in the method since then. Could anybody provide advice and/or a detailed protocol?
In my opinion, AVB sepharose affinity chromatography is the easiest way to purify AAV from crude lysates.
http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/de/GELifeSciences/28411201
We are using it on a HPLC machine but it can also be without it.
Article A Simplified Baculovirus-AAV Expression Vector System Couple...
In our lab, we do iodixanol followed by a column purification. Type of column depends on the serotype one is isolating. After column we include a concentration step as well- using a concentrator. This gives you a higher conc of viruses in lower volumes-which is important if you are going for in vivo administrations.
One of the postdocs in our lab skipped the column method when she was analysing a lot of vectors. And she got decent results. However, in such a case a lot of attention has to be given to the volumes and needless to say all tests and controls have to be purified the same way.
It also depends on if you want to inject only full particles or a combination of empty and fulls. When making AAV the majority of particles will be empty (usually ~10:1 empty to full ratio). CsCl banding is the best way to separate empty and fulls. AVB sepharose is a quick and easy method to get pure vector, but it will purify all vector particles, both empty and full.
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