boosting expression in stable cell line? - (Dec/23/2008 )

I have *finally* (after trying for about one year) managed to make a stable-inducible cell line expressing my (very toxic) gene of interest. It is using the Invitrogen TREx system, which is analogous to the Tet-On system. So, the cells co-express the gene of interest under a tet operator and the tet repressor gene (which binds the tet operator preventing expression). Add tetracycline/doxycycline and this binds the tet repressor, freeing up expression of the GOI.

yay!

My problem is this... the cells expressing the mutant version of the gene are only expressing at a very low level compared to the WT. Rather than bring expression of the WT protein down, I want to try and *boost* expression of the mutant, since I think it's expressing at such a low level as not to have any noticeable effect.

Does anyone know anything I can use to boost expression? I have optimised the level of tetracycline/dox used. The GOI is under a CMV promoter.

Any suggestions very welcome, after spending a year on it, I'll try anything!!

Many thanks
Teela B

Hi Teela B,
CMV promoter is easily inactivated by CpG methylation. Supposedly the demethylation agent 5' azadeoxycytidine may help.

just talking out of my hat, but is there an unmethylatable CMV promoter? I mean a CMV promoter that have been sequence manipulated to prevent methylation.