Hi,
i am doing Luciferase Assay for CCL17-Luc reporter. i am co-transfecting cells with plasmid containg my gene that actually upregulate CCl17 activity. Unfortunatly i am getting high values of Luciferase activity in the group containg only CCL17-Luc plasmid compared to group containg CCL17-Luc and other plasmid containing my gene of interest.
need suggestions about this
Hi Kashif,
High values may be o.k. when your endogenous gene is expressed in your cell line. You may use an unstable variant of luciferase to avoid accumulation of the luciferase protein (see Promega.com) or check other cells for transfection.
That you do not get any activation is more disturbing. Here are a few suggestions:
- Are your sure that your luciferase construct include the response element of your gene of interest? You may have to add more surrounding sequences.
- Are your sure that overexpression of your gene of interest works? You may have to detect the protein by Western blotting.
- Has anyone already used your gene of interest in a luciferase assay? You may request that luciferase construct as positive control.
@uwe Borgmeyer......... about your suggestions....... first and last.... YES.. we got from another group and they published data as well and that CCL17-Luc works. actually i have data from my Real Time and i got up-regulation of CCL17 in response to my another gene that i co-transfected with this construct.
@daniel..... (plasmid1=pCDA3 with my gene that upregulated CCL17) (plasmid2= CCL17-Luc) you ask me about growth/survival of cell. actually i found that in wells containing CCL17-Luc, the cells dying and growth is also restricted. i also co-transfected plasmid 1 while optimizing co-transfection with GFP and i did not found any cell death or cell proliferation. i just found only in wells with plasmid1 and plasmid 2 co-transfected wells
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