We are trying to purify GFP-AAV8 virus from AAV-293 cells, and the GFP is under the TBG promoter in the trans-plasmid. After transfection, green cells are observed under the scope. My questions are, is the GFP expression the leaky expression from LTR? If GFP-cells are only 20%, does that mean the transfection efficiency is low? or is it a combination of transfection efficiency and leaky expression?
I have not worked with the TBG promoter, but using other liver specific promoters (like hAAT), it is very hard to get expression in 293 cells. Are you sure that the green cells you are seeing are actually expressing GFP or could it be auto fluorescence? Also, it is very difficult to get expression from AAV8 vectors in vitro, unless you use a chemical such as doxarubicin or a topoisomerase inhibitor to increase expression.
I would assume that everything is fine and go ahead with the vector purification. You really won't know how the production went until you get a titer (genome) on the vector. You can then test for expression either using a hepatocyte cell line or just try injecting i.v. into a mouse and look for expression in the liver.
Good luck,
John
Thanks so much for the suggestions, John!
The green signal should not be auto fluorescence, since the intensity is relatively strong and is different between cells. I was thinking it might come from the leaky expression of the trans-plasmid by LTR, rather than the AAV8 vector expression, what do you think?
I'm working on the purification and looking forward to know the titer.
That is possible. To be honest we don't often make vector with GFP, so I really wouldn't know if you would expect to see expression. Leaky expression might make sense during the production phase, but I know for sure that AAV8 and liver specific promoters do a very poor job of transducing in vitro.
Are you sure those cells which you see are positive for GFP?
I doubt it because:
AAV8 almost does not infect cell lines ( and in some lines like Huh with very low efficiency). We have tried AAV8-EGFP infection of upto 25000 MOI in some cell lines and still we could not observe any positive cells.
I would also check the mock wells and see if any seemingly positive cells are observed. A false positive might show if the cells have poor viability as dead cells can be very autofluorescent. You can try a PCR based or western analysis to check if the construct is leaky.
Try infecting Huh cell line at high MOI for AAV8. If you feel the construct has leaky expression, try packaging in AAV2/AAV1 capsid and check in common cell lines. I agree with Jonathan. The best way to check AAV8 vector is in vivo analysis. You can inject as low as 10^10 vector and observe fluorescence in liver tissues in a week or two.
Thanks all for the reply.
Have you ever tried primary hepatocytes? I assume it's better than the cell lines. I was hoping to validate the virus in vitro before the in vivo analysis.
Another thing is that, how do you measure the viral titer? Do I need to measure both the vector genome and vector particle titer? I saw kits available for both and wonder if that's what I should use.
I guess I can do all the validation I want, and the key is to do the in vivo injection:)
I have not tried primary hepatocytes, so cannot comment on the utility of the same.
We quantiate vector genome copies by DNA slot blot. It'a a normal DNA blot. Google: Arun Srivastava, JOve. You can see the protocol. Its an old technique which is still accepted by journals. I am sure in vivo injections will happen, once the initial issues are ironed out.
Best
Nishanth
We usually measure our genome titer by qPCR and particle titer using SDS-PAGE and a BSA standard. For your experiment, a genome titer (either by qPCR or slot blot) should be sufficient for your in vivo study.
Good luck!
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