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How to find the mechanism on differential gene expression

Genetics as it applies to evolution, molecular biology, and medical aspects.

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How to find the mechanism on differential gene expression

Postby redhawk » Tue Feb 09, 2010 9:19 pm

We have been doing research on the differential expression of one gene between two cell lines (A and B cell lines), with high expression in A cell line and no expression in B cell line. We found that ~250 bp in the promoter of the gene has its activity in A cell line, but no activity in B cell line. So we think the differential profiling of transcriptional factors between A band B cell lines decide whether the gene is expressed. However, I don’t know to find the transcriptional factors regulating the expression of the gene. Please give me some suggestions.
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Postby JackBean » Tue Feb 09, 2010 9:25 pm

If you wish to find them in silico, look to DNA chips with some knock-outs and stuff.
If you wish to find out practically, you can do ChIP-chip or some kind of affinity chromatography using your DNA as bait
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re:

Postby redhawk » Wed Feb 10, 2010 3:55 pm

JackBean wrote:If you wish to find them in silico, look to DNA chips with some knock-outs and stuff.
If you wish to find out practically, you can do ChIP-chip or some kind of affinity chromatography using your DNA as bait


ChiP-on-chip is used to find the DNA binding sequence with one known transcriptional factor as bait. Hovever, I do not kown transcriptional factors involved in the regulation of the gene expression, If is ChiP-chip suitable for my project?
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Postby JackBean » Wed Feb 10, 2010 9:22 pm

ChIP-chip is for protein-protein interaction.

Anyway, you can try what jonmoulton suggested or some kind of affinity chromatography or anything
http://www.biolib.cz/en/main/

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Re:

Postby redhawk » Wed Feb 10, 2010 10:44 pm

JackBean wrote:ChIP-chip is for protein-protein interaction.

Anyway, you can try what jonmoulton suggested or some kind of affinity chromatography or anything


I'm sorry I don't understand "ChIP-chip is for protein-protein interaction", because I searched that ChIP-chip is for DNA-protein interaction. Please tell me the detail procedure of the application of ChiP-chip in my project". Thanks.
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Postby kolean » Thu Feb 11, 2010 3:08 am

Basically, you can take your 250 bp promoter (plus a few more bp) gene and affix it to some stationary form (beads or an affinity column), and incubate it with lysates. There are many kits out there for you to use for this. If their is a transcription factor it will bind to the promoter part of your gene, and then you will have to carefully 'wash' the stuff away. If done carefully, you should get the main transcription factor and possibly any secondary factors also.
Also, did you look into if your gene expresses mRNA not only in cell line A, but also in cell line B? as oppose to just a protein product in both cell lines. If you see mRNA in both cell lines, but only protein product in one, then I would suggest you look into miRNA regulation of mRNAs.
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Re: How to find the mechanism on differential gene expression

Postby Brent1 » Fri Feb 12, 2010 4:39 pm

Interesting paper recently in Nature along these lines. Very heavy on the bioinformatics, but a cool approach...

Carro MS, Lim WK, Alvarez MJ et al. (2010) The transcriptional network for mesenchymal transformation of brain tumours. Nature 463: 318-325.

Disclosure: Employed by a developer of life science tools. All comments and views expressed are my own and do not necessarily represent those of my of employer.
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