Isotopic Assays for Reporter Gene Activity

Robert E. Kingston1, Jen Sheen1, David Moore2

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 2 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 6.4
DOI:  10.1002/0471141755.ph0604s05
Online Posting Date:  May, 2001
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Abstract

This unit describes two widely used reporter systems that are based on radioactive detection assays. The first assay uses chloramphenicol acetyltransferase (CAT) activity as a measure of the level of expression of a transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group from acetyl CoA to chloramphenicol. Transfected cells are harvested and lysed, and then acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and modified derivatives of the antibiotic are separated from the starting material. The second reporter system uses a kit to perform a simple two‐site radioimmunoassay to quantitate the amount of human growth hormone (hGH) secreted into culture medium by transfected cells. Medium is incubated with 125I‐labeled antibody specific for hGH, and immune complexes are collected by an avidin‐coated bead. The quantity of hormone is determined based on comparison with a standard curve.

     
 
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Table of Contents

  • Basic Protocol 1: Chromatographic Assay for CAT Activity
  • Alternate Protocol 1: In Situ Lysis of Cells for CAT Assay
  • Alternate Protocol 2: Phase‐extraction Assay for CAT Activity
  • Basic Protocol 2: Radioimmunoassay for Human Growth Hormone
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Chromatographic Assay for CAT Activity

  Materials
  • Cells transfected with CAT expression plasmid (Fig. ), in 100‐mm petri plates
  • Phosphate‐buffered saline (PBS)
  • TEN solution (see recipe)
  • 1 M and ice‐cold 0.25 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • 200 µCi/ml [14C]chloramphenicol (35 to 55 mCi/mmol)
  • 4 mM acetyl CoA (store ≤2 weeks at −20°C)
  • Ethyl acetate
  • 19:1 (v/v) chloroform/methanol
  • Rubber policeman or equivalent
  • Thin‐layer chromatography (TLC) tank
  • Whatman 3MM filter paper
  • Thin‐layer chromatography (TLC) sheets (plastic‐backed, silica gel 1B; J.T. Baker)
  • Pen or marker with radioactive ink

Alternate Protocol 1: In Situ Lysis of Cells for CAT Assay

  • Cells transfected with CAT expression plasmid, in 60‐mm petri plates
  • Hypotonic buffer (see recipe)
  • Triton lysis buffer (see recipe)

Alternate Protocol 2: Phase‐extraction Assay for CAT Activity

  • Mammalian cells transfected with CAT expression plasmid or protoplasts transfected with CAT expression plasmid
  • 0.01 µCi/µl [3H]chloramphenicol solution (see step )
  • 5 mg/ml butyryl CoA (store ≤4 months at −20°C)
  • 100 mg/ml unlabeled chloramphenicol
  • 2 M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 2:1 (v/v) tetramethylpentadecane (TMPD)/xylenes

Basic Protocol 2: Radioimmunoassay for Human Growth Hormone

  Materials
  • Cells transfected with hGH expression plasmid (see Fig. )
  • Human Growth Hormone Radioimmunoassay Kit (Allegro Human Growth Hormone, Nichols Institute Diagnostics) containing:
    • 125I‐labeled antibody solution
    • Wash solution
    • Human growth hormone (hGH) standards
    • Avidin‐coated beads
  • 12 × 75–mm round‐bottom test tube
  • γ counter
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Figures

Videos

Literature Cited

Literature Cited
   DeNoto, F.M., Moore, D.D., and Goodman, H.M. 1981. Human growth hormone DNA sequence and mRNA structure: Possible alternative splicing. Nucl. Acids Res. 9:3719‐3730.
   Gilman, M.Z., Wilson, R.N., and Weinberg, R.A. 1986. Multiple protein‐binding sites in the 5′‐flanking region regulate c‐fos expression. Mol. Cell. Biol. 6:4305‐4316.
   Gorman, C.M., Moffat, L.F., and Howard, B.H. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044‐1051.
   Prost, E. and Moore, D.D. 1986. CAT vectors for analysis of eukaryotic promoters and enhancers. Gene 45:107‐111.
   Seed, B. and Sheen, J.‐Y. 1988. A simple phase‐extraction assay for chloramphenicol acetyltransferase activity. Gene 67:271‐277.
   Selden, R.F., Burke‐Howie, K., Rowe, M.E., Goodman, H.M., and Moore, D.D. 1986. Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol. Cell. Biol. 6:3173‐3179.
Key References
   Gorman et al., 1982. See above.
  Describes the initial CAT vectors and the harvest and chromatographic assay.
   Seed and Sheen 1988. See above.
  Describes the phase‐extraction protocol for CAT assays.
   Selden et al., 1986. See above.
  Describes the hGH assay, its use to characterize regulatory events, and vectors that can be used in the assay system.
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