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A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators

  • Wensheng XieEmail author
  • Robert Silvers
  • Michael Ouellette
  • Zining Wu
  • Quinn Lu
  • Hu Li
  • Kathleen Gallagher
  • Kathy Johnson
  • Monica Montoute
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1439)

Abstract

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.

Key words

Cell-based assay Dual luciferase reporter gene assay Firefly luciferase Renilla luciferase High-throughput screening Hit triage 

Notes

Acknowledgments

We thank our colleagues at the Department of Sample Management Technologies for library compound handling and dispensing. We also thank Andrew Benowitz, Ricardo Macarron, Jeff Gross, and Gordon McIntyre for their supervision and leadership.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Wensheng Xie
    • 1
    Email author
  • Robert Silvers
    • 2
  • Michael Ouellette
    • 2
  • Zining Wu
    • 2
  • Quinn Lu
    • 1
  • Hu Li
    • 2
  • Kathleen Gallagher
    • 1
  • Kathy Johnson
    • 1
  • Monica Montoute
    • 2
  1. 1.Department of Target & Pathway Validation, Target SciencesGlaxoSmithKlineCollegevilleUSA
  2. 2.Department of Biological Sciences, Platform Technology and ScienceGlaxoSmithKlineCollegevilleUSA

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