Regarding the infection, we did a scarification in the region before to apply a solution
containing the virus (Takasaki I, Andoh T, Shiraki K, Kuraishi Y
(2000a) Allodynia and hyperalgesia induced by herpes simplex virus type-1 infection in
mice. Pain 86:95–101). We already tried the injection into the hindpaw and the
mouse also developed mechanical hypersensitivity but we did not see any skin lesion.
Thanks @thicunha. AAV works well and I use it when I can. But I don’t have an AAV with an shRNA to the gene I’m targeting. So I’m restricted to using siRNA. Intrathecal is much easier and faster than intraspinal viral injections. I’ve seen people do this before and your recent results with the plasmid and JetPEI make me think siRNA will work pretty well too.
We are currently using i.t. injections of JetPEI with siRNA to target DRG neurons. Our test of a single injection with a fluorescent siRNA showed the transfection of only DRG neurons (~40-70%) and no cells in the spinal cord!
Alex: You may need multiple injections to also target spinal cord cells or use other transfection agents. We tested various agents in our lab and most of them work great in vitro, but very poorly in vivo - sigh!
@achamess I have look again in our data and @tberta is right. Whereas we have 200X increase (compared with
the background) in GFP expression in the DRGs we only see 3X in the spinal cord. Another point, we have injected (it)
5 consecutive days.
Thanks @thicunha and @tberta. This helps me out a lot. I might not waste my time.
@thicunha: In your case, you used a plasmid containing shRNA vs naked siRNA. @tberta: In your case, you did a single siRNA injection.
This protocol from Rohini Kuner’s lab (who always does excellent work, IMO), suggests siRNAs can knockdown spinal cell gene expression (it’s modest though). I may still try multi-day naked siRNA, but your experiences diminish my enthusiasm. I’ll let you know how it goes. Thank you for sharing so openly. This kind of discussion is exactly why this Pain Researcher site was created!