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 Molecular Biology
Unit Number:  Unit 9.7B
DOI:  10.1002/0471142727.mb0907bs29
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes two nonisotopic systems for reporter gene activity in cells transfected with the firefly luciferase expression plasmid or the β‐galactosidase expression plasmid. Both of these chemiluminescent assays have the advantages of high sensitivity and broad linear range. In the chemiluminescent detection procedures given in this unit, both luciferase activity and β‐galactosidase activity can be measured with either a luminometer or a scintillation counter.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Firefly Luciferase Reporter Gene Assay
  • Alternate Protocol 1: Luciferase Assay in Freeze‐Thaw‐Lysed Cells
  • Basic Protocol 2: Chemiluminescent β‐Galactosidase Reporter Gene Assay
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Firefly Luciferase Reporter Gene Assay

  Materials
  • Cells transfected with luciferase expression plasmid (units 9.1 9.4 & 16.13; Fig. 9.7), in 60‐mm petri plates
  • PBS ( appendix 22), ice‐cold
  • recipeTriton/glycylglycine lysis buffer (see recipe)
  • recipeLuciferase assay buffer (see recipe)
  • recipeLuciferin stock solution (see recipe)
  • 25 mM glycylglycine, pH 7.8 (free base, crystalline; Sigma)
  • Firefly luciferase of known activity (Sigma) for use as standard
  • Rubber policeman or equivalent
  • Luminometer (manual or automated) with printer or chart recorder and cuvettes

Alternate Protocol 1: Luciferase Assay in Freeze‐Thaw‐Lysed Cells

  • recipeExtraction buffer (see recipe)
  • Additional reagents and equipment for freeze‐thaw lysis of cells as for chromatographic CAT assay (unit 9.7)

Basic Protocol 2: Chemiluminescent β‐Galactosidase Reporter Gene Assay

  Materials
  • Cells transfected with a β‐galactosidase expression plasmid (units 9.1 9.4 & 16.13; Fig. 9.7), in 60‐mm petri plates
  • Mock‐transfected control cells, in 60‐mm petri plates
  • PBS ( appendix 22)
  • recipeTriton lysis solution (see recipe)
  • β‐galactosidase reaction buffer (see recipe)
  • recipeLight‐emission accelerator solution (see recipe)
  • Rubber policeman (or equivalent)
  • Luminometer with chart recorder and tubes or scintillation counter
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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.
   Brasier, A.R., Tate, J.E., and Habener, J.F. 1989. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. BioTechniques 7:1116‐1122.
   Bronstein, I., Edwards, B., and Voyta, J.C. 1989. 1,2‐Dioxetanes: Novel chemiluminescent enzyme substrates. J. Biolum. Chemilum. 4:99‐111.
   Bronstein, I., Fortin, J.J., Voyta, J.C., Juo, R.R., Edwards, B., Olesen, C.E.M., Lijam, N., and Kricka, L.J. 1994. Chemiluminescent reporter gene assays: Sensitive detection of the GUS and SEAP gene products. BioTechniques 17:172‐178.
   de Wet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R., and Subramani, S. 1987. Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell. Biol. 7:725‐737.
   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.
   Nordeen, S.K. 1988. Luciferase reporter gene vectors for analysis of promoters and enhancers. BioTechniques 6:454‐457.
   Ron, D., Brasier, A.R., Wright, K.A., Tate, J.E., and Habener, J.F. 1990. An inducible 50 kilodalton NFB‐like protein and a constitutive protein both bind the acute‐phase response element of the angiotensinogen gene. Mol. Cell. Biol. 10:1023‐1032.
   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.
   Williams, T.M., Buerlein, J.E., Ogden, S., Kricka, L.J., and Kant, J.A. 1989. Advantages of the firefly luciferase as a reporter gene: Application to the interleukin‐2 gene promoter. Anal. Biochem. 176:28‐32.
   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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library