Hi Experts,
Can you give me an advice on these critical conditions for stereotaxic injection in mice?
1. What is the best age to inject brain regions - striatum and substantia nigra? Based on my readings, 4-8 weeks are the most common weeks. What are the risks of injecting too early? too late?
2. What is the best time to perfuse and analyze the highest expression of an AAV-based target gene?
3. If I don't see any expression are there other things to consider besides AAV quality?
Thanks in advance!
In my experience:
1. I usually wait until mice are around 1.5 months old for injections. Although I have done as young as 1 month. Mice that are too young may yield inaccurate injections.
2. I usually wait 3 weeks for viral expression before doing behaviors or perfusion. I've heard some people wait up to 7 weeks to get good expression for tracing images.
3. Assuming you're using a well known, constitutive promoter, I think the next biggest issue would be clogging of the micropipette, resulting in no virus delivered. This happens to me quite frequently, so now I mark the virus level with a sharpie before injection to ensure the desired volume was injected.
Hi Maxwell,
I appreciate your help and information. Have you had any experience with injecting and comparing CMV/CAG-mediated or GFAP-mediated gene expression for stereotaxic brain injections? I think CMV/CAG-controlled proteins (of course) has better expression regardless of age. I say this because I've recently tried comparing a 4-5 week old and a 18-week old mice and the expression seems to be very consistent and really high. But what I am interested is for genes controlled by the GFAP promoter? Any experience with that?
I've used the CAG promoter and that's worked very well like you've mentioned. But I don't have any experience using the GFAP promoter, unfortunately.
I usually use AAV9 or AAV-DJ for neurons, but not sure what is most common for gial cells. Changing the serotype could be one way to improve expression?