Regarding methylation and luciferase assay - Regarding methylation and luciferase assay (Jun/14/2010 )
I have to do a luciferase based reporter assay with the GFAP promoter and over express Nf1a. Nf1a activates GFAP promoter. Recently I read that it activates by GFAp expression by dissociating Dnmt1 from the promter. If that is the case do I need to methylate my GFAP promoter-luciferase plasmid before transfection to actually see an activation when Nf1a is overexpressed or is it true that plasmids transfected do get de novo methylated in cell lines when kept for long hours. Any ideas on the experiment would be gladly welcome.
Thanks in advance
Yes, foreign sequences such as plasmid transfected into a cells may be methylated through a host defense mechanism. I believe this process happens pretty slowly (not in hours).
Hey thanks pcrman.......would 48 hours be enough for the methylation to happen.
pcrman on Jun 18 2010, 10:39 AM said:
No I don't think anything will happen in 48 hrs.
Hello, there are something I don't understand. I think that if you methylate your GFAP promoter berfore transfection, then Dnmt1 won't combine to the promoter (right?) , so Nf1a does not need to dissociate Dnmt1 from the promoter, which means that it has no use even overexpressed. Finally, the promoter is still methylated and the expression of reporter gene would not be raised significantly. So, what i mean is that since Nf1a does not help remove methyl but dissociate Dnmt1 from the promoter, the promoter should not be methylated before transfection and actually only when it is being methylated in cell, the Nf1a can function and prevent the promoter from being methylated, therefore raising gene expression.. would that be right?