You construct a reporter gene for the Wnt pathway consisting of a promoter to wh

Question

You construct a reporter gene for the Wnt pathway consisting of a promoter to which -catenin can bind, upstream of a series of MS2 repeats and a lacZ gene sequence. You integrate this reporter at a single locus in the genome of the mammalian cell line, and co-express MCB-GFP in these cells. You then treat these cells with either a control solution orWnt ligand, and record the following fluorescence intensities in nuclear spots. In both cases, 100% of cells respond as indicated. From these plots, what can you conclude about how the -catenin controls PolII activity on the promoter binds to? Without directly testing by FISH, what would you estimate is the fold difference in total reporter mRNAbetween the conditions?

Explanation / Answer

In presence of control solution the fluorescence increased with time from 0 to 15 minutes but in presence of Wnt ligand the fluorescence intensity hiked at 5 minutes very quickly and reached at a saturation point at 10 minutes. The GFP protein is attached with the promoter as a reporter gene which means with the active transcription events the expression level of GFP increases and thus the intensity. During control experiment due to administration of control solution the basal level of expression in expression of the promoter containing increased a very little and slowly where as in presence of the specific ligand Wnt receptor activated which causes the stabilisation of -catenin and thus it translocates to the nucleolus and binds with the specific promoter region and induce the transcription with the help of RNA pol III. This transcription rate is reach at maximum level after 10m minutes of Wnt ligand induction and signal continues up to 20 minutes. Then the signal lost in both the cases and thus the transcription stops so the fluorescence intensity decreases.

A qRT PCR can helps to estimate the relative fold change difference between these two conditions. Primer made against the GFP mRNA helps to measure a relative amount of expression of GFP mRNA in cell in both conditions. From where it can be calculated out the relative fold change in mRNA expression.

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