In article <sk.1155349480A at 22.214.171.124>, sk at lilly.com says...
>>We've been trying to express GFP in mammalian cell lines with very
>success. We've used both vectors from Clonetech (pGFP-N1) and a vector
>constructed here with comparable lack of success. The cell lines tried
>HeLa, 293, NRKE, all with no success. A different collegue has said
>sees expression in MCF7 cells, but I haven't tried these yet. I've
>scuttlebutt about these sorts of problems elsewhere. Does anyone have
>feel for what (if any) are the barriers to expression in mammalian
>I've heard that codon usage may be a problem. Any experience with this?
>haven't done Southerns and Northerns yet, but I have no reason to think
>these vectors won't fire in the cell types listed above. Any
>would be greatly appreciated.
>SK at LILLY.COM
I also had little success with the clontec plasmid, however, I've
obtained the mutated GFP which works in primary Sertoli cells and a
Sertoli cell line developed in our lab. This mutated GFP has a Ser65 to
Thr mutation and was described by Helm, Cubitt, and Tslen in a Nature
paper. The protein is supose to be ~6 fold more fluorescent. I don't
know if our lack of success with the clontec plasmid was due to a low
limit of detection but this mutated GFP is easily identified.