Receptor Reporter Systems

David J. Burns1

1 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 6.2
DOI:  10.1002/0471141755.ph0602s00
Online Posting Date:  May, 2001
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Receptor‐linked reporter systems allow the study of pharmacological properties of known receptors, identification of new receptor subtypes, examination of the specific signal transduction pathways linked to receptor activation, and development of functional assays. Reporter systems provide high‐throughput screening methods that allow researchers to expeditiously identify and characterize compounds that modulate the activities of therapeutically relevant receptors or influence events downstream of receptor activation. This unit provides two representative protocols for coupling cell‐surface receptors to reporter molecules that signal receptor activation. The first is a luciferase‐based assay and the second uses green fluorescent protein (GFP). This unit provides a comparison of several reporter assays as well as reporter systems that rely on chloramphenicol acetyltransferase activity.

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Table of Contents

  • Basic Protocol 1: Luciferase Reporter Assay
  • Basic Protocol 2: Green Fluorescent Protein (GFP) Reporter Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Luciferase Reporter Assay

  • TNF‐α–sensitive cell line (e.g., human embryonic kidney 293 cells)
  • Recombinant plasmid: TNF‐α–inducible promoter and puromycin resistance gene subcloned into pGL3 vector containing enhanced luciferase gene (Promega)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.25% (w/v) trypsin/1 mM EDTA in PBS (trypsin/EDTA solution)
  • DMEM‐10 with 1 µg/ml puromycin
  • TNF‐α (Endogen)
  • Reporter lysis buffer (Promega)
  • Luciferase assay reagent (Promega)
  • Recombinant luciferase (Molecular Probes)
  • 175‐cm2 tissue culture flask
  • 50‐ml centrifuge tubes
  • Hemacytometer
  • 96‐well microtiter plate (e.g., VWR Microfluor U‐bottom plates)
  • Luminometer (e.g., Wallac Victor for manual addition of luciferase assay reagent or Labsystems Luminoskan for automatic injection of reagent)

Basic Protocol 2: Green Fluorescent Protein (GFP) Reporter Assay

  • TNF‐α‐sensitive cell line (e.g., human embryonic kidney 293 cells)
  • Recombinant plasmid: TNF‐α‐inducible promoter subcloned into pEGFP vector containing enhanced green fluorescent protein (GFP) gene and neomycin resistance gene (Clontech)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.25% (w/v) trypsin/1 mM EDTA in recipePBS (trypsin/EDTA solution)
  • DMEM‐10 with 1 mg/ml geneticin (G‐418)
  • TNF‐α (Endogen)
  • 175‐cm2 tissue culture flask
  • 50‐ml centrifuge tubes
  • Hemacytometer
  • 96‐well microtiter plate (e.g., Packard white, clear‐bottom tissue culture–treated plates)
  • Fluorometer (e.g., Wallac Victor or Molecular Dynamics Biolumin 960)
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Literature Cited

Literature Cited
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