How to analyze data from a reporter assay
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If Egr-1 is a transcription factor that regulates expression of the GalK gene, then it would be expected that the GalK gene would possess upstream promoter regions to which Egr-1 would bind. Analysis of the published sequence of the GalK gene promoter region revealed that it does indeed contain such regions, along with other potential transcription factor binding sites. To investigate the ability of Egr-1 binding to the Egr-1 promoter to stimulate transcription of the GalK gene, fragments of different lengths of the promoter region were cloned upstream of the firefly luciferase reporter gene into a plasmid vector (pGL-3). Expression of this reporter gene results in a fluorescent signal that can be measured. The resulting plasmids were then introduced (by transfection) into Hep3B cells, which express Egr-1. Cell extracts were later assayed for luciferase activity. The fragments used and the results are shown in Figure 2.
1.How many regions that bind Egr-1 are present in each of the different length GalK promoter fragments?
2.Describe the differences in the relative luciferase activity in the cells transfected with the promoter fragments of different lengths compared with that in cells transfected with the control plasmid (pGL3-b).
3.Considering the data from the (-155) and (-83) fragments only, in comparison to the control, do the data support a role for Egr-1 as stimulating transcription of the GalK gene? Explain your answer.
4.What might the data from the cells receiving the (-272) and (-155) fragments suggest about the possible nature of the transcriptional control of Nkx-2.5 on GalK transcription?
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Solution Summary
The luciferase transcription reporter assay confuse you is determined. In this solution we go in depth to uncover how to best analyze data that we gain from this experiment using a specific question as a guide.
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1) pGK-425, 4 Egr-1 elements
-272, 4 Egr-1
-155, 4 Egr-1
-83, 2 Egr-1
2) Compared to control, -425 showed a modest increase in luciferase intensity, with a slight improvement in the activity with the removal of AP-4 (-272). Removal of Nkx-2.5 (-155) created a ...
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