http://www.ncbi.nlm.nih.gov/pubmed/21775964
My experiment to visualize dendritic spine is according to this article and it successed.
However, recently I am doubting its mechanism. The vector I used is simply pEGFP-C1. And I use lipofactamine 2000 to transfect primary cortical neuron.
Firstly, how does the plasmid go into nucleus? It can only be transcripted into RNA in nucleus I think.
Secondly, if plamids have been transcripted into RNA, how can it diffuse into every dendritic spine? Maybe the distance is too long, some RNA/protein can arrive, while some others may not. It seems there's not location information in the plasmid sequence to direct translocation.
I guess what I observed is part of total spines.
How to deal with this problem?
Hi there,
Whatever the efficiency of GFP diffusion within the dentritic cell and even if protein distribution is uneven, fluoresence signal is used there to define the shape of the cell. Why do you wonder about GFP diffusion whereas there are a lot of endogenous proteins which are exactly in the same situation (ie devoided of any localisation signal)?
Dear Yang,
Interesting and good question. The GFP method have been widely used but I can not recall a study that addressed your question. I regularly fill neurons with dyes and look at spines and filopodia. From my experience what I find is that there is a resistance at the base of the spine limiting amount of dye transfer to the spine itself. The larger the molecule (dye) harder or more limited transfer of the dye into the spines. Therefore with your GFP expression it is possible that you are observing all the spines. See my following paper for a discussion.
best wishes, Refik
Article Emerging Roles of Filopodia and Dendritic Spines in Motoneur...
Hi Refil,
Dendritic spine will restrict the access of HRP(44kDa).
The enhanced GFP I used is 32.7 kDa, much close to 44 kDa. I am not so confident that it can pass all the spine, especially some small ones
Dear Yang,
To find out what percentage of spines you are observing in your GFP expressing cells, you will need to fill your cells with the smallest dye (Neurobiotin or biocytin). This will allow you to quantify and compare the number of spines with GFP and Neurobiotin in the same neuron and its dendrites. I would expect to see variable results with GFP among batches of neurons, but always significantly higher numbers with Neurobiotin. I have developed Neurobiotin-electroporation method that is simple and reliably repeatable for dye-filling neurons in retinal whole-mounts, cultures and brain-slices (see below for details of the method). I would be very interested to see/hear about your results.
best wishes, Refik
Article Semi-loose seal Neurobiotin electroporation for combined str...
Article Neurobiotin Electroporation for Combined Structural and Func...
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