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Functional Screening of Core Promoter Activity

  • Dan Y. Even
  • Adi Kedmi
  • Diana Ideses
  • Tamar Juven-GershonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1651)

Abstract

The core promoter is the DNA sequence that recruits the basal transcription machinery and directs accurate initiation of transcription. It is an active contributor to gene expression that can be rationally designed to manipulate the levels of expression. Core promoter function can be analyzed using different experimental approaches. Here, we describe the qualitative and quantitative analysis of engineered core promoter functions using the EGFP reporter gene that is driven by distinct core promoters. Expression plasmids are transfected into different mammalian cell lines, and the resulting fluorescence is monitored by live cell imaging, as well as by flow cytometry. In order to verify that the transcriptional activity of the examined core promoters is indeed a function of their activity, as opposed to differences in DNA uptake, real-time quantitative PCR analysis is performed. Importantly, the described methodology for functional screening of core promoter activity has enabled the analysis of engineered potent core promoters for extended time periods.

Key words

Engineered promoters Core promoter elements Reporter gene analysis FACS analysis Live cell imaging 

Notes

Acknowledgments

The authors wish to thank Anna Sloutskin for the critical reading of this manuscript.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Dan Y. Even
    • 1
  • Adi Kedmi
    • 1
  • Diana Ideses
    • 1
  • Tamar Juven-Gershon
    • 1
    Email author
  1. 1.The Mina and Everard Goodman Faculty of Life SciencesBar-Ilan UniversityRamat-GanIsrael

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