Target gene regulation by transiently transfected PR - (Mar/09/2009 )

Dear all,

For some time now I am trying to study the function of progesterone receptor in (PR-negative) mammary cell lines.

For this purpose the cells are transiently transfected with 10ng PR construct (in pCl-neo vector) using Lipofectamine 2000. When co-transfected with MMTV-luc or PRE2-luc cells nicely respond to progesterone as measured by luciferase assay. However, at the level of PR target genes (measured by QPCR) nothing is hapening. I've checked multiple known target genes and tested different cell lines, but they all behave the same.

Does anybody have an idea what might be the problem here and how to get the cells 'talking'?

Many thanks in advance.

Grasko on Mar 9 2009, 05:39 AM said:

Strange. I do similar experiments with the glucocorticoid receptor and see results for both luciferase and endogenous genes with 10 ng. Do you have primer sets for luciferase? If so, you can test your qRT-PCR by checking for an increase in luciferase mRNA in duplicate samples for those in which you test for luciferase activity. This would confirm that your qRT-PCR is working ok. Also, what is your positive control? Are you testing gene expression known to be altered by added PR in these cell lines?

Dr Teeth on Mar 9 2009, 03:00 PM said:

I didn't try this approach yet... As the transfection/treatment for gene expression and the luciferase assay are always together within the same experiment I assumed that the luciferase activity is the positive control for both the transfection efficiency and progesterone treatment. The last few experiments were done with T47D subline and the tested target genes are indeed picked based on earlier studies on this cell line. Did you see any major effect of different types of FCS/FBS (I'm currenty using commercial charcoal stripped FCS), different time points or transiently vs stably transfected GR?

Grasko on Mar 9 2009, 10:45 AM said:

When I said, "positive control," I meant for an endogenous gene, but I see that you are using genes known to be regulated in these cell lines, so that might not be important. Regarding using the luciferase as a positive control for transfection efficiency etc., this might not be best. When you perform a luciferase assay, the result you measure is only from transfected cells coexpressing the protein of interest and reporter plasmids. In contrast, for endogenous genes, when you measure the output by qRT-PCR, you will be detecting mRNA levels for both transfected and non-transfected cell types, so often times the "impact" on gene regulation is lower than with reporters. Using stably integrated PR should prevent that problem since this would be a clonal population. For my system, I get larger effects with luciferase reporters than with endogeonus genes during transient transfections due to transfection efficiency. Time points are also important based on the rate of induction/repression for your given gene and whether it is a primary effect, though I assume you are using PR dose times as reported in other literature for those target gene sets. I haven't seen any differences with FBS.

Dr Teeth on Mar 9 2009, 04:17 PM said:

Good point! In that case I can also just check for the PR expression levels in transfected cells (don't have primers for the luciferase)... Thanks