Neuron transfection method-need the best protocol - (Mar/23/2011 )
Topic says its all. I need to transfect mouse cerebellar and/or hippocampal neurons with plasmid or siRNA. What works best with highest survival rate? I have tried Amaxa electroporation-neurons look ugly and don't survive. When I tried AMaxa with plasmid, neurons survived for 3 days and died. I try with Lipofectamine 2000 and see that during 4 hours of incubation with Lipofectamine, many neurons die and processes of remaining neurons get disintegrated. Moreover efficiency of transfection is close to 1% contra promised 25-20% . Please help me if you have a protocol!!!! Thank you!
katenkak on Wed Mar 23 09:43:59 2011 said:
Topic says its all. I need to transfect mouse cerebellar and/or hippocampal neurons with plasmid or siRNA. What works best with highest survival rate? I have tried Amaxa electroporation-neurons look ugly and don't survive. When I tried AMaxa with plasmid, neurons survived for 3 days and died. I try with Lipofectamine 2000 and see that during 4 hours of incubation with Lipofectamine, many neurons die and processes of remaining neurons get disintegrated. Moreover efficiency of transfection is close to 1% contra promised 25-20% . Please help me if you have a protocol!!!! Thank you!
You could try a viral vector such as Adeno associated virus (serotype 9) or lentivirus.
1) Do you know a best vector for lentiviral expression in neurons?
2) I try to find a non-viral protocol, since according to local rules, I can't take live cells outsinde og class 2 area, but only fixed. I need live neurons for real-time measurements.
You may try to find sth. from this paper.
http://www.nature.com/ncb/journal/v11/n6/abs/ncb1881.html
Have you tried Calcium-phosphate method? It's tedious and time-consuming but it does give descent results.
~Labrat612