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

Abstract

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.
OriginalsprogEngelsk
TitelColorectal Cancer : Methods and Protocols
RedaktørerJean-François Beaulieu
Udgivelses stedNew York
ForlagSpringer
Publikationsdato27 mar. 2018
Sider57-77
ISBN (Trykt)978-1-4939-7765-9
DOI
StatusUdgivet - 27 mar. 2018
NavnMethods in Molecular Biology
Vol/bind1765
ISSN1064-3745

Emneord

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

Citer dette

Dahlgaard, K., & Troelsen, J. (2018). Identification and Functional Analysis of Gene Regulatory Sequences Interacting with Colorectal Tumor Suppressors. I J-F. Beaulieu (red.), Colorectal Cancer: Methods and Protocols (s. 57-77). Springer. Methods in Molecular Biology, Bind. 1765 https://doi.org/10.1007/978-1-4939-7765-9_4