Hey folks,
I was hoping someone out there could point me in the direction of a good review / dummies guide to expressing plasmids in neurons? I have a construct that I'd like to express in neurons (it woks fine in HEK cells but the CMV promotor is unhelpful for my needs), so need some guidance on things like Kozak sequences, number of nucleotides between promotor and the start codon, etc.
I also have a rookie question: will plasmids designed as helper plasmid for AAV/lentivirus expressions systems also express well enough using in utero electroporation or lipofectamine / nucleofection transfection of primary neurons? Or will I need a different backbone?
Apologies if these are dumb questions: I'm an electrophysiologist by nature, but I am reasonably comfortable growing plasmids, using restriction enzymes, doing ligations, etc.
Thank you in advance!
Mick
I Michael,
For expression in culture neurons, plasmids with CMV and CAG promoters work very well. If you want to express your protein specifically in neurons I would recomend Tubulin-alpha-1 promoter (some plasmid at addgene has it, you can get it from there). I've also used plasmids with these promotors to drive expression neurons by in utero electroporation and they worked (plenty of expression after 2 days E13.5>15.5).
It will be good to know whether you want to express your protein alone or as a fusion to a tag (GFP, myc, his, etc), since most the plasmids intended to label the N-terminus of your protein have already kosak sequenses flanking the start codon of the tag. On the other hand if you want to express you protein untagged or with a tag to its C-terminus you'll most likely have to insert a kosak sequence before the start codon. probably 90% of the plasmid I use has the GCCACCATG type kosak consensus signal (being ATG the start codon of your protein of interest. Regarding space between promotor and start codon, it is prety flexible. I have ORF linked immediately after the promotor and some starting 150 bp after the promotor and all work fine. However a linker of 15-40 bp would be an average. The linker issue becomes more important for fusion-proteins. If the tag is big and the linker too short it may interfere with the proper folding of the protein you want to express.
In general any plasmid with a functional (neuronal-suitable) expression cassete -promotor-ORF-termination signal- will express in neurons and in neuron-associated assays like IUE. As a rule of thumb, the smaller the plasmid, the more efficient will be the transfection rate.
Hope this helps to initiate, and when you have a more complete idea of the plasmid system fitting your experimental design it will be easier to advise you for the fine tunning of it.
Cheers,
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