GFP expression in mammalian cells

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In article <sk.1155349480A at 40.33.1.1>, sk at lilly.com says...
>
>We've been trying to express GFP in mammalian cell lines with very 
little
>success.  We've used both vectors from Clonetech (pGFP-N1) and a vector
>constructed here with comparable lack of success.  The cell lines tried 
were
>HeLa, 293, NRKE, all with no success.  A different collegue has said 
that he
>sees expression in MCF7 cells, but I haven't tried these yet.  I've 
heard
>scuttlebutt about these sorts of problems elsewhere.  Does anyone have 
any
>feel for what (if any) are the barriers to expression in mammalian 
cells? 
>I've heard that codon usage may be a problem.  Any experience with this? 
 We
>haven't done Southerns and Northerns yet, but I have no reason to think 
that
>these vectors won't fire in the cell types listed above.  Any 
suggestions
>would be greatly appreciated.
>
>Stu Kuhstoss
>SK at LILLY.COM

Stu,

	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.

good luck,

Kerry