luciferase assay - (Feb/07/2008 )

Hi guys,

I am doing luciferase assay in order to confirm that the construct I made, works. So, I have generated a tandem array of DNA sequence which contains binding sites for thyroid hormone receptor and is enhancer element for a promoter. The construct has 48 times of a 35bp-sequence. This sequence is inserted into a luciferase vector. In addition, I have a second construct which has only 1 time of the 35bp sequence and inserted into the luciferase vector, as well. The luciferase activity is dependent on thyroid hormone receptor but indpendent on thyroid hormone. I use HeLa cells in 12-well plates for transient transfection (Fugene6 1:3 ratio). The 1. days I plate cells (50000 cells/well). The 2. day I transfect cells with the luciferase construct + thryroid hormone receptor (different amounts) + pUC18 as filling to 0.5 ug in total. The 3. day I starv cells in 0.5% charcoal treated serum and add thyroid hormone later in the same day. The 4. day I do the luciferase assay.

I expect to see high luciferase activity in the absence of hormone (that I see). I expect to see increasing luciferase activity (in the absence of hormone) propotional to the amount of the receptor construct (that I observe as well). I would also expect an increased luciferase activity in the 1. construct I made. This has the array with many binding sites for the thyroid receptor, which activates transcription. However, when I calculate the luciferase activity/ug DNA, the construct with many binding sites has at least 10 times lower activity then that with only 1 binding site. And this does not make sense to me. The construct I made has more binding sites, so it should give more luciferase activity. That`s the purpose of making the construct.

Has anyone an idea what it could be?
I would be thankful for any help

Hi Zek,
there´s one thing I don´t understand. You wrote "when I calculate the luciferase activity/ug DNA", did you mean luciferase activity/ug protein???.

Hi zek,
I suppose that you made the measure of the luciferase activity in cells untreated with the hormone (is it right?) so your problem seems to be independent of the hormone's actions (I mean differences of proliferation between the transfected cells, I don't really know). Maybe the reporter plasmids you made have different transfecton efficiencies or even the reporter with the 48-times could be competing with the THR expression vector (if you're cotransfecting them) and you really have less THR in these cells and, thus, less luciferase activity. Do you have any control of the THR expression in the transfected cells?? (maybe a western analysis).

Sorry but I think haven't been of help to you. Anyway, good luck!