Hello,
I would like to know which of these two methods (BAC or ZFN) for obtaining for example, a stable GFP-tubulin expressing cell line is better and gives cells that are more similar to physiological WT levels of protein expression?
I appreciate every answer or reference to a reliable source of information
Thank you
I'm not sure what the goal of your experiment is? Usually you use immunofluorescence (fluorescent antibody staining) to visualize tubulin... in which case you can look at the levels of your naturally expressed tubulin...
To answer your question: Neither! Simply use a GFP-tubulin mammalian expression plasmid (such as N1 - prob can find a GFP-tubulin N1 plasmid on Addgene) with a selective marker such as neomycin. Clone in your tubulin promoter (if the plasmid has a different one) - Google is your friend for finding info on the promoter or NCBI or http://www.ensembl.org/index.html for sequences.
If you want to be fancy, you could use an inducible promoter or a degradation tag in addition to your tubulin promoter to regulate expression of your protein.
Then you transfect this plasmid into cells and select cell clones with integrated plasmid using the selective marker. The problem is that you still have the normal tubulin expressed in the cells, BUT I don't think there are viable tubulin knockout cells (don't quote me on this, but fairly sure that tubulin is essential).
Thank you for your answer, I will look into it.
The goal of the experiment is to do live fluorescent microscopy to study dynamics of mitotic spindle assembly. That's why I need cells that have a stable expression of GFP-tubulin among others.
Yes, if you want to do live cell imaging, then you'll have to use a fluorescent protein.
Does it have to be particular cells or will pretty much any cell line do? Because in the latter case, I'd contact the authors of this paper and see if I could get some of the cells they used:
http://www.ncbi.nlm.nih.gov/pubmed/18481305 Live-cell analysis of mitotic spindle formation in taxol-treated cells.
App they used a GFP-tubulin expression plasmid from Invitrogen...
Even if you can't use those cells, the paper should help you understand how to make the cell line.
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