I am very interested by the recently developed SHORT-LIVED EGFP mutant
(Clontech) and would like to know if, once synthesis is prevented by
whatever way, the decrease in fluorescence is rapidly clear-cut
(microscope and FACS).
I'm thinking of using this d2EGFP to test the activity of various
promoters in human myogenic cells. It seems that various common
promoters are somehow shut down for a few days right at differentation
of myogenic cells. Or could it be a phenomenon of "transition-linked
increased mRNA instability" ? (sharing of similar observations, ideas
and comments are very welcome!)
We have been slightly fooled til now using EGFP because the protein is
so stable. If the promoter becomes unfunctional and/or the mRNA becomes
unstable for 48 hours, you wouldn't notice it, until you test for the
activity of your other protein of interest. Testing for the activities
of our proteins of interest appears far more tedious than testing for
d2EGFP at the FACS.
So I'd like to hear from investigators that have used this new variant.
With best regards and thanks in advance.
Physiology Dept. / University of Geneva Medical School