I’m needing to inject viral vectors directly into mouse DRG so that I can localize the expression just to the DRG. I can get very good expression when I inject intrathecally, but that enables the spread of the virus to parts of the CNS where I don’t want it to go.
So I need to do an intra-DRG injection. I know it can be done. A recent paper by Vicuna et al. in Nat. Med used intraganglionic injection to control transgene expression. And I know it can be done in the rat, but for mouse, I don’t have a detailed protocol.
I know that I will need to expose a DRG (lumbar, L4 probably), which will require some kind of laminectomy or small bone removal. It’d be great if anyone has some in-house protocols with images/video to help me perform this procedure. Also, if you’ve injected virus, some info on the volume and titer would be great.
Raquel, my postdoc, will start this kind of experiment in the next weeks and I am sure that she will share any information that may help you.
Our starting point is to replicate (more or less) what it was done by the same group in their previous publication: “Virions were diluted 1∶2 with 20% mannitol and injected unilaterally into L3 and L4 DRGs (1 µl per DRG, or approx. 107 transfection units per DRG)”. There is an impressive image of the GFP expression 2 weeks after injection.
To get to the DRGs, we will use the same approach that it use for the SNL model. We will do some test and try to take a movie of it.
Hopefully, we will become all expert in intraganglionic injections in few months
Thanks @tberta. Looking forward to what you find. We’re going to try as well using a small dental drill to create an open trajectory to the DRG. I’ll try to take videos/pictures.
Any progress on this? I am trying to do DREADD in DRG neurons using the Nav1.8-cre mouse line. The problem is how to get the AAV vector to the DRG. In my case, intrathecal injection may work because Nav1.8 is specific of sensory neurons. However, we have not been successful in delivering the AAV vector intrathecally to mice.
Some labs do intra-DRG injections, but it’s a delicate technique that I myself have not mastered. But if you just want to hit DRG, it’s not necessary, unless you really just want a particular DRG (say, left L5 DRG).
Intrathecal works very well, but it is dependent on titer and serotype and promoter.
Many labs have found that AAV9 in the 10^13 vg/ml range (10 ul typical dose) works very well intrathecally. I’ve done this myself. Which DRGs do you want to hit?
Here is the original reference that I used for my own work:
Recently, this paper found that you can use AAV9 intraplantar and get good retrograde transduction. The key is < P5 animals. I haven’t done this myself although I want.
I’d do the intrathecal route. You need high titer AAV9 though. I’ve had the best success with AAV from Penn vector core. Addgene distributes their stocks now and makes their vectors. It looks like Addgene carries Cre-dependent inhibitory (hm4di) DREADD in AAV9 but not the activating ones. So you may need a custom prep. Make sure it’s 10^13 vg/ml. I haven’t had success with less. See the reference above too. The issue here though is the hSyn promoter. It works, but not amazingly in DRG. CAG is better.
Thanks a lot! I have almost given up on this. We will try a control vector to see if we can get reporter gene expression in the DRG. Then we will be able to order the custom vector using a gene from addgene. I’ll read through the info you provided and come back to you.
If you’re feeling adventurous and want broad dissemination of your AAV in the PNS (not just DRG), you may try the PHP.S serotype from the Gradinaru lab at Caltech. Addgene also offers this. You need to do an intravenous injection. Intrathecal would probably also work but you won’t get the wide spread as IV, I imagine.
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.
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.
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?
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.
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.
Just jumping in here for a second and sort of in relation to my previous post. Have you ever heard or seen or done an intrathecal injection of an AAV construct with any neuron specific promoter which then goes to the DRG and then travels along peripheral nerves to the periphery via anterograde transport?
In theory this should/could happen but obviously in practice things can turn out much differently. I have an idea to try it but was wondering if you had any thoughts?
Sure. IT injection of AAV9 is very effective. Where the transgene goes depends on what the transgene is. But any standard fluorescent protein will go out to the periphery. May take longer but it occurs. ChR2 is membrane localized and looks especially nice. the virus itself probably doesnt make it all the way out to the periphery at high numbers but that’s not what you’re looking for. You want your transgene to get out there after it’s been produced at the soma. So yes, what you’re asking is the default behavior, in my experience. But some genes will be better than others. Also promoters matter. hSyn works in DRG but not amazingly. Might be better to use a Cre mouse and inject a Cre-dependent virus using CAG promoter
Ok great thanks. Cre mouse with Cre-dependent CAG promoter might be the best way to go but we don’t have those mice so likely will try hSyn or PGP9.5 with AAV9 first.
The top 2 panels (A and B) replicate in my lab the findings of this paper:
Schuster, D J, J A Dykstra, M S Riedl, K F Kitto, L R Belur, R S McIvor, R P Elde, C A Fairbanks, L Vulchanova. Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse. Frontiers in neuroanatomy 8: 42-42 (2014). PMC4051274
Note that the construct uses the CB7 promoter and AAV9. We didn’t examine the periphery but got nice staining with eGFP of the axons going into the dorsal horn (panel B)
For panels C and D we used mice expressing Cre in CRF-expressing neurons. CRF expression is poor in DRG, but we had these mice so we tried them. We saw expression of mCherry in a few DRG neurons, consistent with the reported expression of CRF. Many spinal cord neurons also expressed mCherry, presumably because these are the neurons that express CRF.
Thanks. I saw this paper and was asking myself did they check in the periphery. I’ve used mCherry to good effect before so will likely go with that again
If you have a bit of money you could buy peripherin-Cre or advillin-Cre mice from JAX and then inject them with an AAV9 hSyn DIO vector from Addgene. Depends on what you want to do.
Thanks for the info @nikhayes. I know there is interest in these new serotypes of the PHP variety. We have had previous discussion here about variability. Maybe the new ones are better.
I’m currently starting an experimental line in which I need to infect DRGs of some Cre mice, and I cannot express how thankful I am for this entire thread
