Amplification Using CHO Cell Expression Vectors

Robert E. Kingston1, Randal J. Kaufman2, C.R. Bebbington3, M.R. Rolfe3

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 2 University of Michigan, Ann Arbor, Michigan, 3 Celltech Ltd., Slough, United Kingdom
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.23
DOI:  10.1002/0471142727.mb1623s60
Online Posting Date:  November, 2002
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The ability to select for integration of plasmid DNA into the host chromosome allows the generation of stably transfected cell lines. With transfection of a selectable marker linked to a nonselectable target gene (or by cotransfection of the two unlinked genes), high‐level expression of the desired gene is obtained by selecting for amplification of the selectable marker. This unit presents two systems for gene amplification and expression. The first describes the dihydrofolate reductase (DHFR) selection system while the second is based on selection of the glutamine synthetase (GS) gene. The DHFR system is probably more widely used, and results in very high levels of amplification and expression; however, the DHFR amplification process is lengthy and may require several months to isolate and characterize a stable, amplified line. In contrast, the GS system typically requires only a single round of selection for amplification to achieve maximal expression levels.

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Table of Contents

  • Basic Protocol 1: Amplification Using Dihydrofolate Reductase
  • Alternate Protocol 1: Amplification by Cloning at Each Selective Step
  • Basic Protocol 2: Amplification Using Glutamine Synthetase
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Amplification Using Dihydrofolate Reductase

  • pED (Kaufman et al., ) expressing appropriate cDNA; or pCVSVEII‐DHFR or pAd26SV(A) (Kingston et al., ; Kaufman and Sharp, ) and a separate vector expressing appropriate cDNA
  • CHO DXB11 or CHO DG44 cell lines (available from Lawrence Chasin, Columbia University) or CHO GRA (available from Randal Kaufman, University of Michigan)
  • recipeComplete ADT medium (see recipe)
  • 10% glycerol
  • recipeDialyzed fetal bovine serum (FBS; see recipe)
  • Complete α medium (Life Technologies) with 10% dialyzed FBS
  • Sterile vacuum grease
  • 0.05% trypsin/0.6 mM EDTA in PBS (see appendix 22 for PBS), 37°C
  • 2% methylene blue in 50% ethanol (optional)
  • recipe5 mM methotrexate (see recipe)
  • recipeCloning cylinders (see recipe)
  • Additional reagents and equipment for subcloning (unit 3.16), and either CaPO 4‐mediated transfection (unit 9.1), electroporation (unit 9.3), or liposome‐mediated transfection (unit 9.4)
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise indicated.

Alternate Protocol 1: Amplification by Cloning at Each Selective Step

  • Plasmid vector pEE14 (Celltech)
  • recipeComplete Glasgow modified Eagle medium containing 10% dialyzed FBS (complete GMEM‐10; see recipe)
  • CHO K1 cell line (ATCC #CCL61)
  • 100 mM L‐methionine sulfoximine (MSX; Sigma) prepared in PBS (see appendix 22 for PBS; filter sterilize MSX solution and store in aliquots at −20°C; handle carefully)
  • Additional reagents and equipment for subcloning (unit 3.16), CaPO 4‐mediated transfection and glycerol shock (unit 9.1), and cloning by limiting dilution (unit 11.8)
NOTE: All tissue culture incubations are performed in a humidified, 37°C, 5% CO 2 incubator unless otherwise indicated.
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Literature Cited

Literature Cited
   Alt, F.W., Kellems, R.E., Bertino, J.R., and Schimke, R.T. 1978. Selective multiplication of dihydrofolate reductase genes in methotrexate‐resistant variants of cultured mammalian cells. J. Biol. Chem. 253:1357‐1370.
   Bebbington, C.R. and Hentschel, C.C.G. 1987. The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells. In DNA Cloning, Volume III (D. Glover, ed.). Academic Press, San Diego.
   Christman, J.K., Gerber, M., Price, P.M., Flordellis, C., Edelman, J., and Acs, G. 1982. Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant‐acting gene and cloned viral DNA. Proc. Natl. Acad. Sci. U.S.A. 79:1815‐1819.
   Cockett, M.I., Bebbington, C.R., and Yarranton, G.T. 1990. High‐level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/Technology 8:662‐667.
   Davis, S.J., Ward, H.A., Puklavec, M., Willis, A.C., Williams, A.F., and Barclay, A.N. 1990. High‐level expression in Chinese hamster ovary cells of soluble forms of CD4 T lymphocyte glycoprotein including glycosylation variants. J. Biol. Chem. 265:10410‐10418.
   Harfst, E., Johnstone, A.P., Gout, I., Taylor, A.H., Waterfield, M.D., and Nussey, S.S. 1992. The use of amplifiable high‐expression vector pEE14 to study the interactions of autoantibodies with recombinant human thyro trophin receptor. Mol. Cell Endocrinol. 83:117‐123.
   Hayward, B.E., Hussain, A., Wilson, R.H., Lyons, A., Woodcock, V., McIntosh, B., and Harris, T.J.R. 1986. The cloning and nucleotide sequence of cDNA for an amplified glutamine synthetase gene from the Chinese hamster. Nucl. Acids Res. 14:999‐1008.
   Kaufman, R.J. 1989. Selection and coamplification. Meth. Enzymol. 185:537‐566.
   Kaufman, R.J. 1990. Strategies for expressing high‐level expression in mammalian cells. Technique 2:221‐236.
   Kaufman, R.J. and Sharp, P.A. 1982a. Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. J. Mol. Biol. 159:601‐621.
   Kaufman, R.J. and Sharp, P.A. 1982b. Construction of a modular dihydrofolate reductase cDNA gene: Analysis of signals utilized for efficient expression. Mol. Cell. Biol. 2:1304‐1319.
   Kaufman, R., Davies, M., Wasley, L., and Michnik, D. 1991. Improved vectors for stable expression of foreign genes in mammalian cells by use of internal ribosomal entry site from EMC virus. Nucl. Acids Res. 19:4485‐4490.
   Kingston, R.E., Kaufman, R.J., and Sharp, P.A. 1984. Regulation of transcription of the adenovirus EII promoter by EIa gene products: Absence of sequence specificity. Mol. Cell. Biol. 4:1970‐1977.
   Ringold, G., Dieckman, B., and Lee, F. 1981. Coexpression and amplification of dihydrofolate reductase cDNA and the Escherichia coli XGPRT gene in Chinese hamster ovary cells. J. Mol. Appl. Genet. 1:165‐175.
   Sanders, P.G. and Wilson, R.H. 1984. Amplification and cloning of the Chinese hamster glutamine synthetase gene. EMBO J. 3:65‐71.
   Stephens, P.E. and Cockett, M.I. 1989. The construction of a highly efficient and versatile set of mammalian expression vectors. Nucl. Acids Res. 17:7110.
   Urlaub, G. and Chasin, L.A. 1980. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. U.S.A. 77:4216‐4220.
   Urlaub, G., Kas, E., Carothers, A.M., and Chasin, L.A. 1983. Deletion of the diploid dihydrofolate locus from cultured mammalian cells. Cell 33:405‐412.
   Wigler, M., Silverstein, S., Lee, L., Pellicer, A., Cheng, Y., and Axel, R. 1977. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell 11:223‐232.
   Wurm, F.W., Gwinn, K.A., and Kingston, R.E. 1986. Inducible overproduction of the mouse c‐myc gene in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 83:5414‐5418.
Key References
   Kaufman et al., 1991. See above.
  Describes the construction and application of dicistronic DHFR vectors that allow stable, high‐level expression of inserted cDNAs by selection for methotrexate resistance in both DHFR‐containing and DHFR‐deficient cells.
   Cockett et al., 1990. See above.
  Describes the construction of vectors that provide for high‐level expression using the GS gene‐amplification system.
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