How to characterize a promoter sequence - (Jan/16/2006 )
we want to find the basal and functional promoter of a human gene
but the cells that we want to study in already express it at fairly good levels in cell culture
we have not been able to show transcriptional up-regulation in response to any agent in cell culture
When I transfected a 2.5 KB region putative 5'-flanking region which might contain the promoter we did see reporter activity almost 50-100 fold above basal levels (reporter without any promoter)
Could we use these cells to characterize the basal promoter
thanks..any advice will be greatly appreciated
I couldn't see a reason you shouldn't use the cell to study the promoter. It will be nice if you can find another cell which expresses lower your gene.
What you need to do is to construct reporter vectors with serial deleted promoter sequence. A footprinting analysis may also be necessary by which you will probably find different footprints on the promoter by nuclear extracts from the two cells.
So from these two experiments, you will know how long the core promoter is and where are the binding sites of transcriptional factors that may determine transcriptional activity in different cells.