My final objective is to deliver Gs DREADD to the DRG, in order to increase cAMP levels. There are no ready-made AAV vectors for Gs yet, so we will need to get the plasmid from Addgene and have the vector custom-made. However, what I need right now is to get preliminary data for a grant proposal to show that is feasible to infect DRG
OK. Yes, you’ll need to have that custom made.
Regarding your prelim data needs: Infecting DRGs w/ AAV intrathecally is well-accepted now. I don’t think anyone would doubt you could do it (although the prickly-ness of reviewers can never be fully predicted). If it were a more niche technique, I’d say show it. But if you just want to show you can infect DRGs, get that control vector (GFP or tomato) from Addgene and do it. It will take 2-3 weeks (preferably 3 weeks) to see expression. Keep that in mind
So, to answer your question, we want to target all DRG. Nav1.8-Cre will ensure selective expression in nociceptors. We have the Nav1.8-Cre mice.
So you want DRGs throughout the whole body? Maybe then you should try that new PHP.S vector. But that will hit non-DRGs as well. Alternatively, many labs have injected high titer AAV intraperitoneal at P1 and get wide DRG expression. See this nice paper from Fan Wang’s lab
The paper by the Vulchanova group is very encouraging but, as you said, the issue is whether the hSyn promoter will work as well as the CB7 promoter that they used. What did you get using hSyn in DRG?
See this post. That’s what I get with hSyn GFP. This is a high titer AAV from Penn (10^13). You can see there is nice expression, but many DRGs don’t have infection. I think CB7/CAG/CBh would have much higher expression.
We could buy this vector from Addgene AAV-hSyn-DIO-hM3Dq-mCherry with a AAV9 capside at 10^13 vg. Will it work? Or we could get the same vector used by the Vulchanova lad from Addgene https://www.addgene.org/105542/ But this will not tell us if the DREADD vectors with hSyn promoters will work.
The titer is good. That’s what you want. It will infect, just the hSyn promoter will dampen the expression. See this paper, and also my image in the thread above. So it depends on how much expression you need.
If you want high expression of the DREADD transgene, you may need to:
- Order the plasmid
- Make a new plasmid by cloning the DREADD transgene into an AAV backbone with a different promoter, or oppositely, clone in a CAG/CB7/CBh promoter into the hSyn
- Get a custom prep made of the virus.
You may also look in the literature to see if anyone already made such a plasmid. I imagine they did. You could request it, and then have your vector made.
Good luck.