Nonisotopic Assays for Reporter Gene Activity

Allan R. Brasier1, John J. Fortin2

1 University of Texas, Galveston, Texas, 2 Tropix, Inc., Bedford, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 6.5
DOI:  10.1002/0471141755.ph0605s05
Online Posting Date:  May, 2001
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Abstract

This unit describes two nonisotopic systems for reporter gene activity in cells transfected with the firefly luciferase

     
 
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Table of Contents

  • Basic Protocol 1: Chemiluminescent β‐Galactosidase Reporter Gene Assay
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Chemiluminescent β‐Galactosidase Reporter Gene Assay

  Materials
  • Cells transfected with a β‐galactosidase expression plasmid (Fig. ), in 60‐mm petri plates
  • Mock‐transfected control cells, in 60‐mm petri plates
  • PBS (Life Technologies)
  • Triton lysis solution (see recipe)
  • β‐galactosidase reaction buffer (see recipe)
  • Light‐emission accelerator solution (see recipe)
  • Rubber policeman (or equivalent)
  • Luminometer with chart recorder and tubes or scintillation counter
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Figures

Videos

Literature Cited

Literature Cited
   Alam, J. and Cook, J.L. 1990. Reporter genes: Application to the study of mammalian gene transcription. Anal. Biochem. 188:245‐254.
   Bronstein, I., Edwards, B., and Voyta, J.C. 1989. 1,2‐Dioxetanes: Novel chemiluminescent enzyme substrates. J. Biolum. Chemilum. 4:99‐111.
   Fulton, R. and Van Ness, B. 1993. Luminescent reporter gene assays for luciferase and β‐galactosidase using a liquid scintillation counter. BioTechniques 14(5):762‐763.
   Gould, S.J. and Subramani, S. 1988. Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem. 7:5‐13.
   Jain, V. and Magrath, I. 1991. A chemiluminescent assay for quantitation of β‐galactosidase in the femtogram range: Application to quantitation of β‐galactosidase in lacZ‐transfected cells. Anal. Biochem. 199:119‐124.
   Nelis, H.J. and Van Pouke, S.O. 1993. Comparison of chemiluminogenic, fluorogenic, and chromogenic substrates for the detection of total coliforms in drinking water. Proc the Water Quality Technology Conference (AWWA) 1663‐1673.
   Nguyen, V.T., Morange, M., and Bensuade, O. 1988. Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells. Anal. Biochem. 171:404‐408.
   Shaper, N. Harduin‐Lepers, A., and Shaper, J.H. 1994. Male germ cell expression of murine b 4‐galactosyltransferase. A 796‐base pair genomic region containing two cAMP‐responsive elements (CRE)‐like elements, mediates expression in transgenic mice. J. Biol. Chem. 269:25165‐25171.
   Stanley, P.E. 1992. A survey of more than 90 commercially available luminometers and imaging devices for low‐light measurement of chemiluminescence and bioluminescence, including instruments for manual, automatic, and specialized operations, for HPLC, LC, GLC, and microtiter plates. Part I: Description. J. Biolum. Chemilum. 7:77‐108.
   VanDyke, K. 1985. In Bioluminescence and Chemiluminescence: Instruments and Applications, Vol I (K. VanDyke, ed.) pp. 83‐128. CRC Press, Boca Raton, Fla.
   Young, D.C., Kingsley, S.D. Ryan, K.A., and Dutko, F.J. 1993. Selective inactivation of eukaryotic beta‐galactosidase in assays for inhibitors of HIV‐1 TAT using bacterial beta‐galactosidase as a reporter enzyme. Anal. Biochem. 215:24‐30.
Key Reference
   Jain and Magrath, 1991. See above.
  Describes optimization of the chemiluminescent β‐galactosidase assay.
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