In Article <johnson_lab-2609951955550001 at johnsond3.med.yale.edu>,
johnson_lab at qm.yale.edu (Wang Min) wrote:
>Any help on this would be greatly appreciated...
> pRAY-1 is GFP inserted into pDNA3 (CMV promoter-driven GFP), that I
>obtained from GIBCO/BRL. I transfected transiently pRAY-1 into HeLa cells
>and saw nothing by FACS or fluorescence microscopy (the transfections, as
>measured by a second reporter gene, were good).
> This constuct (pRAY-1) has been used in mammalian neuron cells (I
>think), so the coding sequence (codon usage) should be good enough. Are
>there any "tricks" to get the protein to fluoresce or to detect it?
> Thanks for any advice (or commiseration!)
DAve, you are experiencing what many people have seen. It seems that GFP
works for some and not so well for others. I've been talking to people and
I'd guess the variablity is due to a number of factors. Codon usage may be
a problem. Also, fluorescing GFP seems to be a bit temp. sensitive. Make
sure that your incubator is not above 37. (you might even try a little
lower temp.) I've heard that keeping the minimal amount of media may help
with the oxygen dependent chromophore formation. I've heard that the
brighter mutants work better. I've heard that the CGs in the coding
sequence may cause some problems. All in all, I'd guess that with the curent
versions of GFP, functional expression may be iffy and that success or
failure may be due to any number of seemingly small factors. BEcause of the
potential of this reporter, I expect that we'll be hearing about improved
versions shortly. If you don't want to wait and don't want to put in huge
amounts of work to try to improve expression, I'd guess the the things to
try are incubations at 35 rather than 37 and construction of the brighter
mutant (S65T). Good luck.