Question

Question 2 You construct a reporter gene for the NF-kB pathway consisting of a promoter to which NF-kB 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 a mammalian cell line, and co-express MCB-GFP in these cells. You then treat these cells with either a control solution or TNF-a 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 NF-kB changes Polll activity on the promoter it binds to? Without directly testing by FISH, what would you estimate is the fold difference in total reporter mRNA between the conditions? (In other words, how many times greater would the mRNA expression be upon induction?) (8 points) Wnt ligand 10152025 15 time imin) me (min)

Answer 1

Here, when we are adding TNFalpha to the medium, it activates the transcription factor NFkB. As a result, there is increasing activity of RNA POL III ( induction). Without the transcription factor and activators, there is less fluoroscent intensity.

We can detect the fold change in gene expression by doing q PCR. It will give us the change in expression level of two genes.