TCF luciferase reporter assay for Wnt signaling

[sarigsk]

Hi
I ordered the TOPflash/Fopflash luciferase reporter plasmid kit from Upstate, Millipore, where TOPflash plasmid contains three copies of the wild-type TCF binding site making it responsive to TCF while the FOPflash plasmid contains one mutated incomplete copy of the TCF binding site along with two other wild-type copies that make it unresponsive to TCF and hence this plasmid serves as a negative control. However when I do TCF reporter assay I was getting a lower reading for TOPflash compared to FOPflash in one cell line and I can't think of any reason for that.
I would appreciated if someone could share his knowledge with me in this respect.
Thanks indeed

[zargar.shabir]

I am suffering from the same problem. In few cell lines FOP is giving more reading than TOP. Its take my bone to normalize the condition. If you are able to normalize it please write to me on [Only registered users see links. ]

Thank you
SHabir zargar

[sarigsk]

Actually i contacted the technical support team of the company where i got this kit from and they told me that in the absence of Wnt simulation TCF works as a repressor which could be the reason for that. I think this would only be a problem when there is some kind of leakiness from the construct that allows the expression of luciferase!. I had a look at the publications of Prof Roel Nusse of Stanford Uni who is the owner of the Wnt homepage. It seems that he only uses a similar construct to the TOPflash with more TCF binding sites and cotransfects it with a b-galactosidase construct for normalization. But he never uses FOPflash construct. Personally, i am currently using only TOPflash construct but I treat my wild type cells and their modified decendent cells with different concentrations of exogenous Wnt and compare the two resulting graphs and this is working like a charm as I dont really need to worry about or even use it
Hope these info are of help

[zargar.shabir]

thank you for the information, But will it not be a problem to just take only TOP and not FOP. For control you need to show less readings in FOP. And cell lines I am using have constitutive wnt expression. still I need to activate wnt- pathway or not. FOP is the mutated TOP.
Will it be accepted to take TOP only and not FOP. I will be question at every step here.

I hope you will have good Idea in this regard.
Thank you
Shabir

[sarigsk]

I just want to know what you mean by constitutive Wnt expression. I have measured TCF activity in different cell lines. One of them has a mutated B-catenin which makes the Wnt signaling active and hence I was getting a massive reading for TOP compared to FOP. This cell line was not responsive to exogenous Wnt stimulation as the Wnt signaling was already completely activated as a result of the B-catenin mutation. I was only getting higher reading for FOP compared to TOP when I was using another cell line but this one was responsive to exogenous Wnt-3a stimulation and gave readings for TOP that are proportional to the concentrations of exogenous Wnt-3a. I have modified this cell line by gene silencing of a putative player in Wnt signaling and I was getting much less response of TOP to the addition of exogenous Wnt. I guess, the only problem with FOP construct is the leaky expression of luciferase which will be a problem if you don't have an activated Wnt signaling and hence you will get lower readings for TOP as TCF will work as a repressor on the TOP construct that contains wild-type TCF binding sites but not on the FOP construct that contains mutated TCF binding site. As I told you before, I am not sure how you knew that your cell line has constitutive Wnt signaling (i.e. does it overexpress one of the Wnt ligands?). B-catenin levels sometimes are not an indicator of the activity of Wnt signaling as I have a cell line that expresses high level of activated B-catenin (non-phosphorylated form) and yet didn't give a higher reading for TOP compared to FOP. If you are not sure about the status of Wnt signaling in you cell line, it will be useful to add some exogenous Wnt-3a and see what you get.
Hope these are of help