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Methylation of promoter-luciferase construct - investigate methylation on promoter activity (Dec/20/2006 )

Hello all,

Please can you help.. I am a feeble PhD student in need of advice on DNA methylation.

I want to assess the methylation status of a gene in one cell vs the other. More specifically I want to know if the promoter is silenced via DNA methylation. This gene is silenced (not sure how, think its methylation) in one particular cell and not the other. But when I clone the promoter, its active in both cells (mRNA not expressed in the silenced cell, suggesting transcription is silenced- but how) I think its because my promoter does not have sufficient time in the cell to see any methylation.

What I wanted to do was to artificially methylate all the CpG sites in my promoter and then transfect into both types of cell; do you think this is straighforward? also, as many E-coli methylate plasmids, is this methylation pattern different to mammalian methylation of human DNA sequences?

Please can anyone help! I would be very grateful.

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-shojjahd-

Use the SssI DNA methylase from New England Biolabs. This is very straight forward. If you think the E. cloi methylation is going to affect your results, you can prepare your DNA in dam- dcm- strains.

-tap14-

I once fully methylated a CMV promoter and it almost knocked all the promoter activity out. My feeling is that it is hard for you to mimic the cell methylation pattern in the test tube. The expeiment may not be as meaningful as you think. I could be wrong on this.

-genehunter-1-

True, depends if complete methylation of you promoter is a biologically relevant or if it is normally just partially methylated

-tap14-

I used SssI methylase and I have little control over the degree of methylation in that system. Maybe in the presence of some transcription factors the outcome would be different.

-genehunter-1-

Maybe a dumb question, but have you assessed the promoter methylation in both cell types? Is it different in any CpG? blink.gif

-kr├╝melmonster-

I am doing the exact same thing shojjhad as your experiment,

I am using promega's pGL3 system and I have cloned into the basic vector, my promoter of interest. I have already shown that the control vector is silenced by DNA methylation through SssI methylase (from NEB) now I am attempting to show the same thing for my promoters of interest.

But depending on your system, I suspect it would be mammalian, SssI will mimic CpG methylation. You will run into problems with if you are studying plants for example.

Nick

-methylnick-

Hi Nick, I want to do the same thing but one question needs your advice:

did you use the whole pGL construct for methylation? if then there are quite a lot of CpGs in the construct, how could you exclude the effect by their methylation?

Or, can I just methylate the specific region of promoter then insert it into the vector? but then how can I amplify the contruct? Thanks!


QUOTE (methylnick @ Dec 20 2006, 05:03 PM)
I am doing the exact same thing shojjhad as your experiment,

I am using promega's pGL3 system and I have cloned into the basic vector, my promoter of interest. I have already shown that the control vector is silenced by DNA methylation through SssI methylase (from NEB) now I am attempting to show the same thing for my promoters of interest.

But depending on your system, I suspect it would be mammalian, SssI will mimic CpG methylation. You will run into problems with if you are studying plants for example.

Nick

-Schiwann-