Reporters of Gene Expression: Enzymatic Assays

Matt Lorincz1, Mario Roederer2

1 Fred Hutchinson Cancer Research Center, Seattle, Washington, 2 Stanford University, Stanford, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.5
DOI:  10.1002/0471142956.cy0905s06
Online Posting Date:  May, 2001
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Abstract

Initially developed for use in bacteria, reporter gene systems are now being utilized in eukaryotic cells.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Use of Fluorescein DI‐β‐D‐Galactopyranoside and Flow Cytometry to Quantitate β‐Galactosidase Enzymatic Activity
  • Alternate Protocol 1: Use of Fluorescein DI‐β‐D‐Glucuronide and Flow Cytometry to Quantitate β‐Glucuronidase Enzymatic Activity
  • Support Protocol 1: Use of the MUG or MUGlcU Assay for Independent Determination of β‐GAL or GUS Activity
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Use of Fluorescein DI‐β‐D‐Galactopyranoside and Flow Cytometry to Quantitate β‐Galactosidase Enzymatic Activity

  Materials
  • Single‐cell suspensions of transfected and control cultures
  • Staining medium (see recipe)
  • 2 mM fluorescein di‐β‐D‐galactopyranoside (FDG) working solution (see recipe)
  • Quenching solution (see recipe)
  • 50 mM phenylethyl β‐D‐thiogalactopyranoside (PETG; Sigma; prepare in H 2O and store indefinitely at −20°C)
  • 12 × 75–mm polystyrene tubes (Falcon)
  • Flow cytometer with sample chilling capability and 488‐nm excitation source
  • Additional reagents and equipment for immunophenotyping (unit 6.2)

Alternate Protocol 1: Use of Fluorescein DI‐β‐D‐Glucuronide and Flow Cytometry to Quantitate β‐Glucuronidase Enzymatic Activity

  • 2 mM fluorescein di‐β‐D‐glucuronide (FDGlcU) working solution (see recipe)
  • 250 mM 1,4‐saccharolactone (1,4‐SL; see recipe)

Support Protocol 1: Use of the MUG or MUGlcU Assay for Independent Determination of β‐GAL or GUS Activity

  • Cell cultures: β‐gal‐ or Gus‐transfected and control
  • 0.13% Triton X‐100/Z buffer prepared fresh with 1 part 1% (v/v) Triton X‐100/ recipeZ buffer stock and 7 parts Z buffer
  • 250 mM 1,4‐saccharolactone (1,4‐SL; see recipe)
  • 30 mM 4‐methylumbelliferyl β‐D‐galactopyranoside (MUG) or 30 mM 4‐methylumbelliferyl β‐D‐glucuronide (MUGlcU; see reciperecipes)
  • Z buffer (see recipe)
  • Stop buffer (see recipe)
  • 96‐well flat‐bottom microtiter plate
  • Microtiter plate centrifuge
  • Multichannel pipettor
  • Fluorescence plate reader capable of excitation at 355 nm and emission at 460 nm (e.g., Fluoroskan II, Flow Labs)
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Figures

Videos

Literature Cited

Literature Cited
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Key References
   Fiering et al., 1991. See above.
  Significant new additions to the original protocol and a more quantitative (and biochemical) characterization of the assay.
   Lorincz et al., 1996. See above.
  First detailing of the FACS‐Gus system for mammalian cells.
   Nolan et al., 1988. See above.
  The first description of a flow cytometry–based assay for reporter gene activity.
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