Intraganglionic injections into mouse DRG?


#1

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.

@tberta do you have any thoughts on this?


#2

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 :wink:


#3

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.


#4

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.


#5

Hi @marvizon

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.

https://www.cell.com/cell-reports/pdf/S2211-1247(18)30624-7.pdf

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.

See this discussion

https://www.addgene.org/viral-service/aav-prep/chemogenetics/

Good luck. Let us know how it goes.


#6

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.

Great site, BTW!


#7

Hi @marvizon

Thanks for the kind words. Please tell your colleagues and students about this site! The more people the better.

That is a good plan. I recommend this Cre-dependent tomato vector to test.

https://www.addgene.org/100048/

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.

https://www.addgene.org/28306/


#8

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.

Any thoughts?


#9

@marvizon

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.


#10

You may also try to request this mouse