Can transcriptional regulation research be done in vivo? - No cell line is availbale (Feb/21/2007 )
Dear friends,
I am trying to do some research about the transcriptional regulation of a gene of interest.
The biggest headache is that it was found that the gene's expression decrease rapidly in vitro. So there is now no cell line available for the transient transfection.
So I am thinking if I can use DNase I footprinting assay with 5 400bp-overlapped-sequences that can cover the 1.5 kb 5' flanking promoter region. After got the regions being protected by protein, I can search in the TF database to get some candidate TFs.
At the same time, I wonder if there is a method that can do the transcriptional regulation study in vivo? Because the assay in vitro can't show the real situation in vivo.
Thank you for the help!
You could use those 400pb regions of your promoter and do an yeast one-hybrid assay. Basically it allows you to identify transcription factors that bind to your bait (in this case the promoter region) using yeast as host. The catch is that you need to screen a cDNA library in order to do that, so you either need to make the library, which is kind of time consuming I presume, or borrow one from someone who's already done so. I am doing one of these screens but the cDNA library was already prepared by my colleagues so I didn't have to start from the very begining.
Nevertheless, the in vivo assay should be complemented by an in vitro assay, like gel shift or so, to confirm the interactions.
Good luck on that
If you have in vivo tissue from you fixed for histology (ex vivo now I suppose) , you might be able to do Chromatin-IP and Gel-shift experiments from fixed tissue. Since you sound like you know the promoter sequences interested in, it should be pretty easy to pickout antibodies to candidate TF's. If I find the ref's I was looking at I'll post them later. Cheers!
Thank you for the suggection.
Maybe I didn't explain it clearly.
Now I just use the 1.5 kb 5' flanking sequence to predict the possible transcription factors which can bind to this DNA fragment. Actually, I got 57 conserved predicted TF binding sites and almost 50 predicted transcription factors. So it is almost impossible for me to try them one by one by EMSA or ChIP.
Until now, I don't know the promoter motifs of the gene of interest. I think if it is possible that I divide the 1.5 kb flanking sequence into 8-10 200 bp fragments and being incubated with the nuclear extract from the rat's tissue. So I can detect which part is protected from DNase I by protein in the nuclear extract. Then I can use those protected sequences to check the candidate TFs.
Is my idea practicable?
Did anyone do in this way before.
Thank you very much for the help!
Nevertheless, the in vivo assay should be complemented by an in vitro assay, like gel shift or so, to confirm the interactions.
Good luck on that
Thank you for the suggestion!
Unfortunately, I don't have a cDNA library and there isn't a person that I can borrow it from. Moreover, I dpn't think my boss will allow me to spend several months to set up a cDNA library.
Thank you anyway.
Nobody can help me?
How about this:
Do you know when the gene is normally expressed? If so, you could get hold of the promoter region and mix it with a nuclear extract from cells that should be expressing the protein. That way, you should be able to pull down whatever TFs are interacting with the promoter.
Maybe.
Perhaps.
If you're lucky...
Do you know where this gene is expressed? If it is in the liver hepatocytes, muscle or lung endothelial cells, your life is much easier. because you can transfect these cells with naked DNA or by liposome-DNA complexes.