DEX and GRE - (Oct/16/2006 )

Hi,

I am identifying a GRE in a promoter using luciferase reporter assay. Currently, I have transfected my construct (promoter which contain GRE+PGL3) and induced with Dex. However, there was't any effect on it.

But i doubt my DEX is working probably since it has been stored more than a years in -20 c.

Do u guys know any construct that can serve as my positive control ( sure induce by DEX)? even better that i can amplify that fragment and make the construct myself.


cyk

I used a pGRE.Luc vector from BD Biosciences about 5 years ago. It's just 3 GRE's fused to the minimal TATA like promoter of HSV and drives the expression of luciferase. I was just trying to determine if the endogenous GR was active or not, so I transfected the vector into HeLa cells (+ control) and saw 40-60 fold induction with 100nM and 1uM Dex. In my own experimental cell line, which is less sensitive to Dex, I saw 2-10 fold induction. So, it's a simple reporter vector that you can use as a positive control.

I I remember correctly, Dex powder has a shelf life of 5 years at 4 degrees. In solution it's shelf life is 1 week at 4 degrees and a year at -20. However, I have used it past one year at -20 and it was still fine. Call Sigma, or whatever company it was purchased from and ask them if they have any data after a year storage.