A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.
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.
Department of Target & Pathway Validation, Target Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA. wensheng.2.xie@gsk.com.
Wensheng
W
Xie
Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA.
Robert
R
Silvers
Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA.
Michael
M
Ouellette
Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA.
Zining
Z
Wu
Department of Target & Pathway Validation, Target Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA.
Quinn
Q
Lu
Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA.
Hu
H
Li
Department of Target & Pathway Validation, Target Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA.
Kathleen
K
Gallagher
Department of Target & Pathway Validation, Target Sciences, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA.
Kathy
K
Johnson
Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA.
Monica
M
Montoute
D000818:Animals; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D048888:Fireflies / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics; D049410:Luciferases, Renilla / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D048790:Renilla / Q000235:genetics; D014162:Transfection / Q000379:methods; D015854:Up-Regulation / Q000187:drug effects*; D055545:gamma-Globins / Q000235:genetics*
D055545:gamma-Globins; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly
Cell-based assay; Dual luciferase reporter gene assay; Firefly luciferase; High-throughput screening; Hit triage; Renilla luciferase