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Searching Novel Clock Genes Using RNAi-Based Screening

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Circadian Clocks

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2130))

Abstract

RNA interference (RNAi) allows for the selective downregulation of gene expression by neutralizing targeted mRNA molecules and has frequently been used in high-throughput screening endeavors. Here, we describe a protocol for the highly parallel RNAi-mediated downregulation of gene expression in order to search for components involved in circadian rhythm generation. We use lentiviral gene transfer to deliver shRNA expressing plasmids into circadian reporter cells ensuring for efficient and stable knockdown. Circadian rhythms are monitored using live-cell bioluminescence recording of synchronized reporter cells over several days. In addition, we present a new software tool (ChronoStar) for efficient, parallel time-series analysis to extract rhythm parameters such as period, phase, amplitude, and damping.

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Acknowledgments

We thank Maike Mette-Thaben and Daniel Lohse for excellent technical assistance. Work in AK’s laboratory is supported by the Deutsche Forschungsgemeinschaft (SFB740/D2 and TRR186/A17).

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Author notes

  1. Stephan Lorenzen

    Present address: Bernhard-Nocht-Institut, Hamburg, Germany

Authors and Affiliations

  1. Laboratory of Chronobiology, Charité—Universitätsmedizin Berlin, Berlin, Germany

    Bert Maier, Anna-Marie Finger & Achim Kramer

  2. Institute for Theoretical Biology, Humboldt-Universität zu Berlin, Berlin, Germany

    Stephan Lorenzen & Hanspeter Herzel

Authors
  1. Bert Maier
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  2. Stephan Lorenzen
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  3. Anna-Marie Finger
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  4. Hanspeter Herzel
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  5. Achim Kramer
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Corresponding author

Correspondence to Achim Kramer .

Editor information

Editors and Affiliations

  1. Chronobiology and Sleep Research Group, Institute of Pharmacology and Toxicology, University of Zurich, Zürich, Switzerland

    Steven A. Brown

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Maier, B., Lorenzen, S., Finger, AM., Herzel, H., Kramer, A. (2021). Searching Novel Clock Genes Using RNAi-Based Screening. In: Brown, S.A. (eds) Circadian Clocks. Methods in Molecular Biology, vol 2130. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0381-9_8

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  • DOI: https://doi.org/10.1007/978-1-0716-0381-9_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0380-2

  • Online ISBN: 978-1-0716-0381-9

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