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Functional Screening of Core Promoter Activity

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Book cover Mammalian Synthetic Promoters

Part of the book series: Methods in Molecular Biology ((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.

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Acknowledgments

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

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Authors and Affiliations

  1. The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Life Sciences Building (212) Room 102, Ramat-Gan, Israel

    Dan Y. Even, Adi Kedmi, Diana Ideses & Tamar Juven-Gershon

Authors
  1. Dan Y. Even
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  2. Adi Kedmi
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  3. Diana Ideses
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  4. Tamar Juven-Gershon
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Corresponding author

Correspondence to Tamar Juven-Gershon .

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Editors and Affiliations

  1. Department of Biochemical Pharmacology, William Harvey Research Institute, Queen Mary University of London, London, UK

    David Gould

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Even, D.Y., Kedmi, A., Ideses, D., Juven-Gershon, T. (2017). Functional Screening of Core Promoter Activity. In: Gould, D. (eds) Mammalian Synthetic Promoters. Methods in Molecular Biology, vol 1651. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-7223-4_7

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  • DOI: https://doi.org/10.1007/978-1-4939-7223-4_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-7221-0

  • Online ISBN: 978-1-4939-7223-4

  • eBook Packages: Springer Protocols

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