Promoter experiments - What's next? (Feb/19/2007 )
I've already been doing a bioinformatic analysis of the region upstream from de first exon of my known sequence that codes for the protein of interest, can anybody tell me what to do next? I mean I'm planning to study the region with a construction having a reporter, but how do I decide which is the hypothetical promoter region? for maybe I don't see activity due to negative regulation.......
I'm not very good at this issue......
Does anyone has some information or protocols? or links where I can find out what to take into account when doing this kind of experiments??
Have you thought about a "dissection" of the promoter region? If you already know the predicted promoter region after exploring with specialized software, you can dissect it choosing several regions of that zone, so if you clone your reporter construction downstream each chosen fragment, you could find where is the promoter region of your gene of interest.
I hope it helps.
Typically the characterization of a poorly described promoter would be carried out using a 5' large sequence with the generation of smaller constructs by 5' deletions in an attempt to map out the functional elements. The region of interest should, if possible extend to the start codon. Since you have carried out some bioinformatics (I assume transcription factor binding sites) you could think about verifying these using EMSA. There are several other techniques that can be used to investigate promoter regions. This link goes over some of the different techniques. It’s focused on polymorphisms, since that is what I'm interested in but the principles should apply to all promoter analysis. http://www.clinsci.org/cs/104/0493/cs1040493.htm.
Our lab uses the pGL3 reporter vector form promega, have a look at their website for more info on that system.