Identification and Functional Analysis of Gene Regulatory Sequences Interacting with Colorectal Tumor Suppressors

Katja Dahlgaard, Jesper Troelsen

Publikation: Bidrag til bog/antologi/rapportBidrag til bog/antologiForskningpeer review


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.
TitelColorectal Cancer : Methods and Protocols
RedaktørerJean-François Beaulieu
UdgivelsesstedNew York
Publikationsdato27 mar. 2018
ISBN (Trykt)978-1-4939-7765-9
StatusUdgivet - 27 mar. 2018
NavnMethods in Molecular Biology


  • CDX2 GPA33 Enhancer Promoter Transcription factor Promoter reporter assay Transfection Luciferase

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