Transcription factors regulation - testing the effect of NFkB on expression (Aug/12/2007 )

Hi all,
I'm studying the promoter of a gene, so far I got the 2000bp long sequence upstream the TSS by PCR, inserted in pGEM T plasmid. I will cut and insert in pCAT vector used for activity assays.
Apart from testing activity in cells (HeLa cells) I also want to see the effect of NFkB on transcription. What should I do to do that?
I know there are several assays such as EMSA and Chip to do this kind of thing but, which should I used?
I also have a tumoral cell line which expresses NFkB constitutively (I suppose since it has a lot of this protein), so I wondered if I could do transfection and activity assays in this cells to compared the results with those got with HeLa cells. Could this work?

Can I test interaction without using nuclear extracts? is there something more simple like having the transcription factor or a plasmid that codes for it, and doing a specific reaction like EMSA with just the TF I'm interested in?? (I don't know if this is possible, maybe it's more difficult, I know NFkB has basically two subunits....so maybe is not as simple as I imagine in my head!!)

Sugestions please!!


biotech

You could search for NFkappaB sites in the promoter sequence with a web-based program like TFSearch. Then mutate them by site-directed mutagenesis in your promoter reporter and look for a loss of activity when you stimulate with IL-1, TNF or LPS (something that activates NFkB).

There are 5 NFkB subunits (p65, p50, p52, cRel, RelB) which can form various homo- or hetero- dimers. Even though your tumour line expresses the subunits you will need to stimulate them to see activity in most cells.

Ceri