Functional Analysis of the Heme Oxygenase‐1 Gene Promoter

Jawed Alam1

1 Alton Ochsner Medical Foundation, New Orleans, Louisiana
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 9.7
DOI:  10.1002/0471140856.tx0907s06
Online Posting Date:  May, 2001
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Abstract

Basal and inducer‐dependent transcription activity of the heme oxygenase‐1 gene promoter can be measured using the luciferase reporter gene in transfection assays. Methods are provided in this unit for purification of plasmid DNA, transient and stable transfection of DNA into cells, and measurement of luciferase and beta‐galactosidase enzyme activities using chemiluminescent‐based assays. These protocols can be used to identify inducer‐activated cis elements.

     
 
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Table of Contents

  • Basic Protocol 1: Transient Transfection of Reporter Gene Constructs into Mammalian Cells and Measurement of Reporter Enzyme Activities
  • Alternate Protocol 1: Stable Transfection of Reporter Gene Constructs into Mammalian Cells
  • Support Protocol 1: Plasmid DNA Purification
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Transient Transfection of Reporter Gene Constructs into Mammalian Cells and Measurement of Reporter Enzyme Activities

  Materials
  • Adherent eukaryotic cells: e.g., HeLa (human cervical epithelial adenocarcinoma), MCF‐7 (human mammary epithelial adenocarcinoma), NIH 3T3 (mouse embryo fibroblast), L929 (mouse connective tissue fibroblast‐like), Hepa (mouse hepatoma), or RAW 264.7 (mouse peritoneal macrophage)
  • Complete medium (depending on cell line used)
  • Plasmid containing luciferase gene under control of ho‐1 promoter (see Table 97.80.4711) purified by CsCl gradient (see protocol 3)
  • Plasmid containing β‐galactosidase gene (see Table 97.80.4711) purified by CsCl gradient (see protocol 3)
  • Carrier CsCl‐purified plasmid DNA (see protocol 3)
  • 3 M sodium acetate, pH 5.2 (see recipe)
  • 95% ethanol
  • 2.5 M CaCl 2 (see recipe)
  • 2× HEPES‐buffered saline (HeBS; see recipe)
  • Glycerol/PBS solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • HEPES‐buffered, serum‐free medium (appropriate to cell line; see recipe) containing inducing agent of interest
  • Cell lysis buffer (see recipe)
  • Luciferase assay buffer (see recipe)
  • 0.2 mM luciferin solution (see recipe)
  • β‐galactosidase chemiluminescent substrate solution (see recipe)
  • Light emission accelerator (Tropix)
  • 6‐well tissue culture plates
  • 14‐ml polystyrene round‐bottom tubes (Falcon)
  • Platform shaker or rocker
  • Luminometer with measuring tube
  • 48°C bath or heating block
  • Additional reagents and equipment for mammalian cell culture ( appendix 3B)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Stable Transfection of Reporter Gene Constructs into Mammalian Cells

  • 20 mg/ml G418 (see recipe)
  • Mammalian expression plasmid encoding neo gene and control expression vector lacking neo gene (e.g., pBSSK)
  • Bicinchoninic acid kit for protein determination (Sigma; optional)
  • 10‐cm tissue culture dishes
  • 12‐ to 96‐well tissue culture plates

Support Protocol 1: Plasmid DNA Purification

  Materials
  • Plasmid‐bearing E.coli colonies on agar plate
  • 2× YT bacteria culture medium (see recipe)
  • Antibiotic (e.g., ampicillin, kanamycin)
  • Glucose solution (see recipe)
  • NaOH/SDS solution (see recipe)
  • Potassium acetate solution (see recipe)
  • Isopropanol
  • TE buffer, pH 8.0 (see recipe)
  • CsCl
  • 10 mg/ml ethidium bromide (see recipe)
  • CsCl/TE buffer solution (see recipe)
  • CsCl/TE buffer–saturated isopropanol (see recipe)
  • 3 M sodium acetate, pH 5.2 (see recipe)
  • 95% or 100% and 70% ethanol
  • Sterile toothpicks
  • Culture tubes
  • 37°C shaker incubator
  • 2‐liter flask
  • 250‐ml ultracentrifuge bottles (for a Sorvall GSA rotor)
  • Sorvall RC‐5B superspeed centrifuge and Sorvall GSA rotor (or equivalent)
  • 10‐ml heat sealable ultracentrifuge tubes
  • Beckman L‐70 ultracentrifuge and Beckman VTi 65 rotor (or equivalent)
  • Stand and clamp
  • 18‐G (or wider) needles
  • 3‐ to 5‐ml sterile syringes
  • 15‐ml screw‐cap polypropylene tubes
  • Sorvall SS‐34‐rotor with rubber adapters (or equivalent)
  • 1.5‐ml microcentrifuge tubes
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Figures

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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.
  Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A.M.K., and Cook, J.L. 1999. Nrf2, a cap 'n' collar transcription factor regulates induction of the heme oxygenase‐1 gene. J. Biol. Chem. 274:26071‐26078.
   Choi, A.M.K. and Alam, J. 1996. Heme oxygenase‐1: Function, regulation, and implication of a novel stress‐inducible protein in oxidant‐induced lung injury. Am. J. Respir. Cell. Mol. Biol. 15:9‐19.
  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.
  Elbirt, K.K., Whitmarsh, A.J., Davis, R.J., and Bonkovsky, H.L. 1998. Mechanism of sodium arsenite–mediated induction of heme oxygenase‐1 in hepatoma cells. J. Biol. Chem. 273:8922‐8931.
  Gorman, C. 1985. High efficiency gene transfer into mammalian cells. In DNA Cloning II—A Practical Approach (D.M. Glover, ed.) pp. 143‐190. IRL Press, Oxford.
  Graham, F.L. and van der Eb, A.J. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456‐467.
  Immenschuh, S., Hinke, V., Ohlmann, A., Gifhorn‐Katz, S., Katz, N., Jungermann, K., and Kietzmann, T. 1998. Transcriptional activation of the haem oxygenase‐1 gene by cGMP via a cAMP response element/activator protein‐1 element in primary cultures of rat hepatocytes. Biochem. J. 334:141‐146.
  Jordan, M., Schallhorn, A., and Wurm, F.M. 1996. Transfecting mammalian cells: Optimization of critical parameters affecting calcium‐phosphate precipitate formation. Nucl. Acids Res. 24:596‐601.
   Kingston, R.E., Chen, C.A., Okayama, H., and Rose, J.K. 1996. Calcium phosphate transfection. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.1.4‐9.1.11. John Wiley & Sons, New York.
  Maines, M.D. 1992. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Boca Raton, Fla.
  Mortensen, R., Chesnut, J.D., Hoeffler, J.P., and Kingston, R.E. 1997. Selection of transfected mammalian cells. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.5.1‐9.5.19. John Wiley & Sons, New York.
   Pellacani, A., Wiesel, P., Sharma, A., Foster, L.C., Huggins, G.S., Yet, S.‐F., and Perrella, M.A. 1998. Induction of heme oxygenase‐1 during endotoxemia is downregulated by transforming growth factor‐β1. Circ. Res. 83:396‐403.
   Rose, J.K. 1996. Optimization of transfection. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.1.1‐9.1.4. John Wiley & Sons, New York.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Takeda, K., Ishizawa, S., Sato, M., Yoshida, T., and Shibahara, S. 1994. Identification of a cis‐acting element that is responsible for cadmium‐mediated induction of the human heme oxygenase gene. J. Biol. Chem. 269:22858‐22867.
Key References
   Alam and Cook, 1990. See .
  Compares advantages and disadvantages of various reporter genes commonly used in mammalian cells.
   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.
  Provides a more detailed characterization of the luciferase assay used herein.
   Graham and van der Eb, 1973. See .
  These two articles examine parameters essential for successful transfection by the calcium phosphate precipitation method.
   Jordan et al., 1996. See .
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