Identification and Functional Analysis of Gene Regulatory Sequences Interacting with Colorectal Tumor Suppressors
Several tumor suppressors possess gene regulatory activity. Here, we describe how promoter and promoter/enhancer reporter assays can be used to characterize a colorectal tumor suppressor proteins’ gene regulatory activity of possible target genes. In the first part, a bioinformatic approach to identify relevant gene regulatory regions of potential target genes is presented. In the second part, it is demonstrated how to prepare and carry out the functional assay.
We explain how to clone the bioinformatically identified gene regulatory regions into luciferase reporter plasmids by the use of the quick and efficient In-Fusion cloning method, and how to carry out transient transfections of Caco-2 colon cancer cells with the produced luciferase reporter plasmids using polyethyleneimine (PEI). A plan describing how to set up and carry out the luciferase expression assay is presented. The luciferase/β-galactosidase (Dual Light) assay presented is a highly sensitive assay that can monitor small changes in the promoter/enhancer activity and includes an internal control monitoring transfection efficiency.
Key wordsCDX2 GPA33 Enhancer Promoter Transcription factor Promoter reporter assay Transfection Luciferase
This work was supported by The Danish Council for Independent Research (4004-00140B). We would like to thank Johanne Davidsen and Sylvester Larsen for reagents and fruitful discussions on the method and Louise Torp Dalgaard for constructive comments.
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