Dear all, I have a question concerning AAV-transduction. I would like to use the AAV System to introduce a KO in NHDF-cells. For this, I transfected HEK293T-cells with 3 different Plasmids (see attached). The viral production seems to work very well(strong GFP/RFP signal). After isolation, I determined the viral titer via qPCR analysis. I calculated a titer of around 1x10^13 vp/ml. But when I try to transduce different cells (HEK, HT1080, NHDF), I detected no fluorescent signal at all. For the transduction, I used different MOI (from 10.000 to 10) but with no success.
Do you have any idea, what I could change? The used plasmids contain the needed components, right?
Best regards and many thanks
Bastian
OK, you say ' The viral production seems to work very well(strong GFP/RFP signal', is that only after transfection? That only means that your promoters work in 293s. I notice you're using the synapsin promoter, that's not a promoter that works very well outside of neurons if I recall correctly. None of your target cells are neuronal, is there a reason you need to use the hSyn promoter? Is this virus for use in vivo or just in cell culture?
Also, to make AAV you need the Adeno genes (pHelper, pladeno5 are examples), a rep/cap serotype plasmid and a plasmid with your transgene of interest with the AAV ITRs. From your diagrams, you seem to have two ITR plasmids and one combined rep/cap + Adeno plasmid. Are you using both ITR plasmids in the same production?
What serotype are you making? If you are making AAV2 and not using a transduction booster (eg etoposide) AND you're using a weak promoter (hSyn), I'm not surprised that you can't see much.
Dear Jill,
thanks a lot for your reply. I want to use the virus just in cell culture. Actually, I just use the hSyn-promoter containing plasmid just primarily as a transfection control. My gRNAs are cloned in both vectors, PX552 and PX601. In both cases, the gRNA expression is controlled by an U6-promoter.
Yes, all the desired Adeno genes are encoded on the pDP2rs vector. Until now, I used both ITR plasmids in the same production. Do you think, that this could be a problem?
Yes, I am making serotype 2 virus. The "key components" (SaCas9, gRNA) are not controlled by the hSyn promoter. So, couldn´t it be, that I don´t see a GFP signal, because of the weak hSyn promoter? Today, I started two different approaches: First: pDP2rs + PX552, Second: pDP2rs + PX601.
In which way is a booster such as etoposide affecting the transduction?
Best regards
Bastian
You may well already know this, but AAV is not usually a good choice for cell culture unless the cells are not dividing, or at least dividing very slowly. You will lose the infection very quickly. If you only need transient expression thought it could be OK. What is your target cell type or cell line?
The issue with using two ITR plasmids in the same production run is it that you might get an uneven ratio of viruses - one might work well and the other might work poorly. Of course you could check this by using primers specific to both viruses (eg primers for GFP and primers for Cas9). If you don't really need the two viruses mixed together then it might be best to do two separate productions at the same time. This will tell you if your transfection protocol is working and you can assess each virus separately.
The human synapsin promoter is very neuron specific as far as I know. After transfection you can usually see plenty of expression but not after transduction. I suggest using B35 cells if you want to check hSyn-driven expression - they're available from the ATCC, very easy to grow and AAV infects them very well.
Etoposide increases AAV transduction by up to 300-fold, and in my experience it makes a huge difference (Article DNA synthesis and topoisomerase inhibitors increase transduc...
) But, it tends to be toxic to cells. So the exposure time has to be very short (hours, not days).Any reason why you're using AAV2? It's the most common/widely used serotype but except in rare cases, it doesn't tend to be the most efficient choice.
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