On treating my hepatocytes (non cancerous) with a hyperlipidemic drug, I expected to induce the CYP1a1 gene, but neither the gene nor the positive control showed any response in a time and dose dependent manner. Are my hepatocytes unresponsive to the drug? How could I prove that the drug has entered the cell? I tried various other positive controls too, without success. Any suggestions?
Hi Aruloli,
could be several explanations - Cyp1a1 is under the regulation of Ahr receptor, so you should try potenet agonists of Ahr (like dioxin), maybe as a positive control to see whethre this type of regulation works in your cells in generally. Are you using primary cells or established cell lines? In some cell lines maybe this gene is silenced via methylation and is not inducible via AhR anymore. Or you have so many natural ligands of AhR in your culture system that it´s already on limit of induction. I would try to overexpress AhR in these cells addtionally to the agonist treatment, maybe that helps.
Best regards,
masha
I'd suggest to go and get your positive controls up and running before testing your gene of interest.
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