sensitive, stable transcriptional markers?

L. Bastian yacman at u.washington.edu
Wed Dec 18 17:30:46 EST 1996


Greeting Earthlings,
	I am interested in measuring activity of a weak promoter in cells
that transfect poorly after a week of post-transfection treatment with a
differentiating agent that I believe might increase promoter activity.  I
know this plan poses significant technical challenges. (Theme from "Rocky"
playing).  I know this promoter can activate expression of luciferase in
transiently transfected cell lines, but luciferase activity is rapidly
lost from the cells in a few days.  Galactosidase activity is relatively
stable (at least in the cell line) and is still detectable a week
after transfection.  Unfortunately, I can only detect activity using the
very strong CMV promoter.  Galactosidase is not sensitive enough, at least
in my hands, for my weak promoter of interest.  My guess is that CAT is
sensitive enough but very likely, like luciferase, to be lost from the
cells.  So.  Questions.  Has anyone directly compared the secreted
alkaline phosphatase and growth hormone markers in terms of utility and
sensitivity? Better yet, any suggestions for boosting my Gal sensitivity?
Any other suggestions?   Also, while I'm at it, why the heck _is_ the sky
blue? :). 


L. Scot Bastian Ph.D.			206-762-1010 ext. 1918 (voice)
University of Washington at the         206-764-2689 (FAX)
  Veterans' Affairs Medical Center      yacman at u.washington.edu (Email)
M/S 151 
1660 S Columbian Way South 
Seattle WA 98108 





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