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
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Abstract

This unit describes two nonisotopic systems for reporter gene activity in cells transfected with the firefly luciferase expression plasmid or the -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 -galactosidase activity can be measured with either a luminometer or a scintillation counter.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Firefly Luciferase Reporter Gene Assay
  • Alternate Protocol: Luciferase Assay in Freeze-Thaw-Lysed Cells
  • Basic Protocol 2: Chemiluminescent -Galactosidase Reporter Gene Assay
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
     
 
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Materials

Basic Protocol 1: Firefly Luciferase Reporter Gene Assay

 Materials
  • Cells transfected with luciferase expression plasmid (UNITS 9.1-9.4 & 16.13; Fig. 9.7B.1), in 60-mm petri plates
  • PBS (APPENDIX 2), ice-cold
  • Triton/glycylglycine lysis buffer (see recipe)
  • Luciferase assay buffer (see recipe)
  • Luciferin 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: Luciferase Assay in Freeze-Thaw-Lysed Cells

 Additional Materials (also see Basic Protocol 1)
  • Extraction buffer (see recipe)
  • Additional reagents and equipment for freeze-thaw lysis of cells as for chromatographic CAT assay (UNIT 9.7A)

Basic Protocol 2: Chemiluminescent -Galactosidase Reporter Gene Assay

 Materials
  • Cells transfected with a -galactosidase expression plasmid (UNITS 9.1-9.4 & 16.13; Fig. 9.7B.2), in 60-mm petri plates
  • Mock-transfected control cells, in 60-mm petri plates
  • PBS (APPENDIX 2)
  • Triton lysis solution (see recipe)
  • -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

  •  FigureFigure 9.7B.1 Luciferase expression vectors. (A) pOLUC is a promoterless vector containing a luciferase gene. It is derived from pGEM3, a high-copy ampicillin-resistance plasmid whose multiple cloning sites are flanked by SP6 promoter/primer sequences (SP6 P/P) and T7 promoter/primer sequences (T7 P/P). Unique restriction sites for insertion of sequences of interest are indicated by asterisks (*). (B) pSV2LUC contains the strong eukaryotic simian virus-40 (SV40) promoter/enhancer sequences inserted into pOLUC for use as a positive control for luciferase expression or as a standard for normalization between transfections. SV40 ENH designates the 72-base-pair repeats of the SV40 enhancer, G+C box(es) are the SP1-binding sites of the SV40 promoter, and EES designates the early-early transcription start sites.
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  •  FigureFigure 9.7B.2 -galactosidase expression vector. p-gal-Basic lacks eukaryotic promoter and enhancer sequences and allows insertion of promoter-containing DNA fragments into the polylinker upstream of the lacZ gene. Enhancer sequences can be cloned into the polylinker or into unique sites downstream of lacZ. All four vectors in this series contain an SV40 intron and SV40 polyadenylation signal downstream of lacZ to ensure proper and efficient processing of the transcript. The vector backbone contains a f1 origin for single-stranded DNA production and a pUC-19 origin of replication and ampicillin-resistance gene for propagation in Escherichia coli. The multiple cloning site (polylinker) region is identical in all four vectors in the series; in p-gal-Promoter and in p-gal-Control a 202-bp promoter-containing fragment has been inserted between Bgl II and Hin dIII. The sequence of p-gal-Basic has been deposited in GenBank (Accession #U13184).
    View Image

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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 -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 -galactosidase in the femtogram range: Application to quantitation of -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 -galactosidase assay.

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