B-catenin pathway - Lef/TCF-sensitive reporter gene assay (Sep/11/2008 )

Hi,

I'm reading papers on the wnt/beta catenin canonical pathway b/c my Phd research would involve b-catein and I'd like to clarify a few thoughts.

Investigators use the Lef/Tcf reporter gene assay to study the effect of silencing a component of teh wnt pathway (e.g. Dvl-1) on Lef/Tcf transcription. Why not just measure the b catenin expression? What's so significant of the Left/Tcf transription. Also, how does the Lef/Tcf reporter work? Does anyone have a link to its diagram?

Thanks

One of the reasons why people use the reporter assay is due to the fact that it is quite easy to quantify the result. If you want to look at b-ctn levels by Western blotting, this is not so quantitative as luciferase values. Additionally, the reporter assay, albeit an artificial method, is a measure for the activation of the pathway. The amount of b-ctn as such is not a guarantee for a high transcriptional activity, it's better to test it directly. A good alternative for this would be to measure the expression levels by qRT-PCR from a direct Wnt target gene (but this is more work than a simple luciferase assay).

I don't have a scheme for the assay, but actually it's just a plasmid with a luciferase reporter gene which is preceded by some TCF/Lef binding site (a.k.a. TOPFLASH), upon binding of TCF/Lef, the reporter gene is transcribed and can be measured.

I hope I have made some things clear, if not just let us know and I'll try again

One of the reasons why people use the reporter assay is due to the fact that it is quite easy to quantify the result. If you want to look at b-ctn levels by Western blotting, this is not so quantitative as luciferase values. Additionally, the reporter assay, albeit an artificial method, is a measure for the activation of the pathway. The amount of b-ctn as such is not a guarantee for a high transcriptional activity, it's better to test it directly. A good alternative for this would be to measure the expression levels by qRT-PCR from a direct Wnt target gene (but this is more work than a simple luciferase assay).

I don't have a scheme for the assay, but actually it's just a plasmid with a luciferase reporter gene which is preceded by some TCF/Lef binding site (a.k.a. TOPFLASH), upon binding of TCF/Lef, the reporter gene is transcribed and can be measured.

I hope I have made some things clear, if not just let us know and I'll try again

beta-catenin also binds to the cadherin complex at the plasma membrane, so you're also seeing this pool if you analyze the total amount of b-ctn. Based on the presence or absence of cell cell adhesion, the amount of b-ctn at the plasma membrane will differ. This b-ctn at the PM is not responsible for transcriptional activation, so you'll get a wrong estimate if you just use b-ctn amounts (although in some cases this may be enough when a luciferase assay isn't feasible, although I would then prefer to look at the levels of nuclear b-ctn).

Thnak you very much for the clarification, truly appreiated.