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

Katja Dahlgaard, Jesper Troelsen

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-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.
Original languageEnglish
Title of host publicationColorectal Cancer : Methods and Protocols
EditorsJean-François Beaulieu
Place of PublicationNew York
PublisherSpringer
Publication date27 Mar 2018
Pages57-77
ISBN (Print)978-1-4939-7765-9
DOIs
Publication statusPublished - 27 Mar 2018
SeriesMethods in Molecular Biology
Volume1765
ISSN1064-3745

Keywords

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

Cite this

Dahlgaard, K., & Troelsen, J. (2018). Identification and Functional Analysis of Gene Regulatory Sequences Interacting with Colorectal Tumor Suppressors. In J-F. Beaulieu (Ed.), Colorectal Cancer: Methods and Protocols (pp. 57-77). New York: Springer. Methods in Molecular Biology, Vol.. 1765 https://doi.org/10.1007/978-1-4939-7765-9_4
Dahlgaard, Katja ; Troelsen, Jesper. / Identification and Functional Analysis of Gene Regulatory Sequences Interacting with Colorectal Tumor Suppressors. Colorectal Cancer: Methods and Protocols. editor / Jean-François Beaulieu. New York : Springer, 2018. pp. 57-77 (Methods in Molecular Biology, Vol. 1765).
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Dahlgaard, K & Troelsen, J 2018, Identification and Functional Analysis of Gene Regulatory Sequences Interacting with Colorectal Tumor Suppressors. in J-F Beaulieu (ed.), Colorectal Cancer: Methods and Protocols. Springer, New York, Methods in Molecular Biology, vol. 1765, pp. 57-77. https://doi.org/10.1007/978-1-4939-7765-9_4

Identification and Functional Analysis of Gene Regulatory Sequences Interacting with Colorectal Tumor Suppressors. / Dahlgaard, Katja; Troelsen, Jesper.

Colorectal Cancer: Methods and Protocols. ed. / Jean-François Beaulieu. New York : Springer, 2018. p. 57-77 (Methods in Molecular Biology, Vol. 1765).

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

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AB - 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.

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