Expression of Cloned Receptors in Mammalian Cell Lines

Murali Gopalakrishnan1, Eduardo J. Molinari1

1 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 6.3
DOI:  10.1002/0471141755.ph0603s00
Online Posting Date:  May, 2001
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Abstract

The expression of cloned receptor and ion‐channel proteins is a very useful tool for studying the physiological and pharmacological properties of receptors. The process of introducing a gene of interest into eukaryotic cells by biochemical or physical methods is termed transfection. After transfection, some of the DNA is transported into the nucleus, where it is transcribed. The resulting mRNA is transferred into the cytoplasm and translated into protein. The transfection method described in this unit is a versatile, plasmid‐based, liposome‐mediated gene‐transfer technique known as lipofection. This technique has gained wide acceptance because of its simplicity, suitability for a wide range of cell types, reasonable transfection efficiencies, and reproducibility. This unit also includes a procedure for assessing transfection efficiency via cotransfection with a reporter gene (lacZ) and assay for coexpression.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Receptor Expression by Cationic Lipid–Mediated Gene Transfer into Mammalian Cells
  • Support Protocol 1: Assessment of Transfection Efficiency by Coexpression of β‐Galactosidase
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Receptor Expression by Cationic Lipid–Mediated Gene Transfer into Mammalian Cells

  Materials
  • Cells (e.g., HEK‐293) growing in tissue culture
  • Dulbecco's phosphate‐buffered saline (D‐PBS; Life Technologies)
  • 0.25% trypsin/1 mM EDTA (Life Technologies)
  • Complete growth medium (see recipe)
  • Plasmid DNA containing gene of interest (see recipe)
  • Serum‐free medium: DMEM (Life Technologies)
  • Low‐serum medium: Opti‐MEM I (Life Technologies)
  • Lipid reagent (e.g., Lipofectamine or Lipofectamine PLUS; Life Technologies)
  • 0.25% (w/v) trypsin/EDTA
  • Complete growth medium (see recipe) containing appropriate antibiotic for selection
  • 35‐ and 100‐mm tissue culture dishes
  • 5‐ to 15‐ml tissue culture tube
  • Polystyrene tubes (e.g., Corning)
  • Tabletop centrifuge
  • Glass cloning cylinders (8 mm high; 6 or 8 mm o.d.; Bellco Glass), autoclaved
  • 24‐well tissue culture plates
NOTE: All culture incubations are performed in a humidified 37°C, 5% (or 10%) CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.

Support Protocol 1: Assessment of Transfection Efficiency by Coexpression of β‐Galactosidase

  Materials
  • Cells transfected with lacZ‐containing plasmid and untransfected cells as control
  • Dulbecco's phosphate‐buffered saline (D‐PBS; Life Technologies)
  • Glutaraldehyde fixative solution (see recipe)
  • Xgal solution (see recipe), freshly prepared
  • Inverted microscope
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Figures

Videos

Literature Cited

Literature Cited
   Ahring, P.K., Strøbæk, D., Christophersen, P., Olesen, S.P., and Johansen, T.E. 1997. Stable expression of the human large‐conductance Ca2+‐activated K+ channel α‐ and β‐subunits in HEK293 cells. FEBS Lett. 415:67‐70.
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 1998. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Bertrand, D., Buisson, B., Krause, R.M., Hu, H.Y., and Bertrand, S. 1997. Electrophysiology: A method to investigate the functional properties of ligand‐gated channels. J. Recept. Signal Transduct.Res. 17:227‐242.
   Besnard, F., Even, Y., Itier, V., Granger, P., Partiseti, M., Avenet, P., Depoortere, H., and Graham, D. 1997. Development of stable cell lines expressing different subtypes of GABAA receptors. J. Recept. Signal Transduct. Res. 17:99‐113.
   Beverley, S.M. 1992. Enzymatic amplification of RNA by PCR. 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. 15.4.1‐15.4.6. John Wiley & Sons, New York.
   Boussif, O., Lezoualc'h, F., Zanta, M.A., Mergny, M.D., Scherman, D., Demeneix, B., and Behr, J.P. 1995. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethyleneimine. Proc. Natl. Acad. Sci. U.S.A. 92:7297‐7301.
   Brewer, C.B. 1994. Cytomegalovirus plasmid vectors for permanent lines of polarized epithelial cells. Methods Cell Biol. 43:233‐245.
   Bronstein, I., Fortin, J., Stanley, P.E., Stewart, G.S., and Kricka, L.J. 1994. Chemiluminescent and bioluminescent reporter gene assays. Anal. Biochem. 219:169‐181.
   Buisson, B., Gopalakrishnan, M., Sullivan, J.P., Arneric, S.P., and Bertrand, D. 1996. Human α4β2 neuronal nicotinic receptor in HEK 293 cells: A patch clamp study. J. Neurosci. 16:7880‐7891.
   Chalfie, M., Tu, Y., Euskirechen, G., Ward, W.W., and Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802‐805.
   Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., and Danielsen, M. 1987. Lipofection: A highly efficient, lipid‐mediated DNA‐transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84:7413‐7417.
   Gopalakrishnan, M., Monteggia, L.M., Anderson, D.J., Molinari, E.J., Piattoni‐Kaplan, M., Donnelly‐Roberts, D., Arneric, S.P., and Sullivan, J.P. 1996. Stable expression, pharmacologic properties and regulation of the human neuronal nicotinic acetylcholine α4β2 receptor. J. Pharmacol. Exp. Ther. 276:289‐297.
   Heim, R. and Tsien, R.Y. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6:178‐182.
   Kahana, J.A. and Silver, P.A. 1996. Use of the A. victoria green fluorescent protein to study protein dynamics in vivo 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. 9.7.22‐9.7.28. John Wiley & Sons, New York.
   Lukas, R.J. and Cullen, M.J. 1988. An isotopic ion efflux assay for the functional characterization of nicotinic acetylcholine receptors. Anal. Biochem. 175:212‐218.
   MacGregor, G.R., Nolan, G.P., Fiering, S., Roederer, M., and Herzenberg, L.A. 1991. Use of E‐coli lacZ (β‐galactosidase) as a reporter gene. In Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols (E.J. Murray and J.M. Walker, eds.) pp. 217‐235. Humana Press, Totowa, N.J.
   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.
   Moss, B., Elroy‐Stein, O., Mizukami, T., Alexander, W.A., and Fuerst, T.R. 1990. Product review: New mammalian expression vectors. Nature 348:91‐92.
   Schroeder, K.S. and Neagle, B. 1996. FLIPR: A new instrument for high throughput optical screening. Biomol. Screening 1:75‐81.
Key References
   Felgner, P.L. 1991. Cationic liposome–mediated transfection with lipofectin reagent. In Methods in Molecular Biology Vol. 7: Gene Transfer and Expression Protocols, (E.J. Murray and J.M. Walker, eds.) pp. 81‐89. Humana Press, Totowa, N.J.
  In addition to lipofection, this book also discusses other DNA transfection and viral infection techniques and certain methods of analysis of stable transfectants.
  Roth, M.G. (ed). 1994. Methods in Cell Biology, Vol. 43: Protein Expression in Animal Cells. Academic Press, San Diego.
  A good compilation covering various expression systems for transgene expression in mammalian cells.
Internet Resources
  http://www.promega.com/promtech/faq
  A good compilation of common questions and answers on transfection and reporter systems.
  http://www.nwfsc.noaa.gov/protocols.html
  A source for methodologies—e.g., in DNA purification and sequencing.
  http://biochem.boehringer.com/prod_inf/biblios/dotaptoc.html
  Bibliography of various cell lines transfected with cationic lipid reagent DOTAP.
  http://www.invitrogen.com/bin/wwwboard/expvectors/
  Mammalian expression forum.
  http://www.lifetech.com/online/tech.html
  Provides a general guide to transfection of eukaryotic cells with cationic lipids.
  http://www.horizonpress.com/gateway/protocols.html
  A resource for certain recombinant DNA and cell biology protocols.
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