Skip to main content
SpringerLink
Log in
Book cover

Protein Expression in Mammalian Cells pp 161–172Cite as

Generation of High-Expressing Cells by Methotrexate Amplification of Destabilized Dihydrofolate Reductase Selection Marker

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 801))

Abstract

A method combining the use of a destabilized dihydrofolate reductase (DHFR) selection marker with methotrexate (MTX) amplification to generate high-expressing cells is described here. The selection marker expression is weakened with the use of the murine ornithine decarboxylase PEST region and AU-rich element to target the DHFR protein and mRNA, respectively, for degradation in the cell. Cells that produce higher levels of DHFR protein, and the adjoining recombinant protein gene, can compensate for the more rapid turnover of the DHFR protein and survive the selection process. This effect can complement MTX amplification to reduce the amount of MTX and shorten the time needed to generate a high-expressing clone. The gene of interest is first inserted into an expression vector that contains a destabilized DHFR selection marker. The resulting expression vector is then linearized and transfected into suspension CHO-DG44 cells. Selection is performed by culturing the cells in a selection medium lacking hypoxanthine and thymidine. Low concentrations of MTX are then used to amplify the transfected genes for increased protein expression. A single cell cloning protocol is also described. This can be used after each stage of MTX amplification to isolate high-expressing clones that are also consistent producers over longer culture periods.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Thomsen DR, Stenberg RM, Goins WF, Stinski MF. (1984) Promoter-regulatory region of the major immediate early gene of human cytomegalovirus. Proc Natl Acad Sci U S A. 81(3):659–63.

    Google Scholar 

  2. Kim CH, Oh Y, Lee TH. (1997) Codon optimization for high-level expression of human erythropoietin (EPO) in mammalian cells. Gene. 199(1–2):293–301.

    Google Scholar 

  3. Kalwy S, Rance J, Young R. (2006) Toward more efficient protein expression: keep the message simple. Mol Biotechnol. 34(2):151–6.

    Google Scholar 

  4. Kaufman, R.J., Sharp, P.A. (1982) Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase cDNA gene. J. Mol Biol. 159:601–621.

    Google Scholar 

  5. Cockett MI, Bebbington CR, Yarranton GT. (1990) High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Biotechnology (N Y). 8(7):662–7.

    Google Scholar 

  6. Chen, L., Xie, Z., Teng, Y., Wang, M., Shi, M., Qian, L., Hu, M., Feng, J., Yang, X., Shen, B., Guo, N. (2004) Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene. J. Immunol. Methods. 295:49–56.

    Google Scholar 

  7. Sautter, K., Enenkel, B. (2005) Selection of high-producing CHO cells using NPT selection marker with reduced enzyme activity. Biotechnol. Bioeng. 89(5):530–538.

    Google Scholar 

  8. Niwa, H., Yamamura, K., Miyazaki, J. (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108:193–200.

    Google Scholar 

  9. Ng SK, Wang DI, Yap MG. (2007) Application of destabilizing sequences on selection marker for improved recombinant protein productivity in CHO-DG44. Metab Eng. 9(3):304–16.

    Google Scholar 

  10. Zubiaga, A.M., Belasco, J.G., Greenberg, M.E. (1995) The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol. Cell. Biol. 15(4):2219–2230.

    Google Scholar 

  11. Ross, J. (1995) mRNA stability in mammalian cells. Microbiol. Rev. 59(3):423–450.

    Google Scholar 

  12. Guhaniyogi, J., Brewer, G. (2001) Regulation of mRNA stability in mammalian cells. Gene 265(1–2):11–23.

    Google Scholar 

  13. Bakheet, T., Frevel, M., Williams, B.R.G., Greer, W., Khabar, K.S.A. (2001) ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins. Nucleic Acids Res. 29(1):246–254.

    Google Scholar 

  14. Bakheet, T., Williams, B.R.G., Khabar, K.S.A. (2003) ARED 2.0: an update of AU-rich element mRNA database. Nucleic Acids Res. 31(1):421–423.

    Google Scholar 

  15. Ghoda, L., van Daalen Wetters, T., Macrae, M., Ascherman, D., Coffino, P. (1989) Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation. Science 243:1493–1495.

    Google Scholar 

  16. Ghoda, L., Sidney, D., Macrae, M., Coffino, P. (1992) Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation. Mol. Cell. Biol. 12(5):2178–2185.

    Google Scholar 

  17. Loetscher, P., Pratt, G., Rechsteiner, M. (1991) The C terminus of mouse ornithine decarboxylase confers rapid degradation on dihydrofolate reductase. J. Biol. Chem. 266(15):11213–11220.

    Google Scholar 

  18. Li, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., Fan, C., Huang, C.C., Kain, S.R. (1998) Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273(52):34970–34975.

    Google Scholar 

  19. Corish, P., Tyler-Smith, C. (1999) Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 12(12):1035–1040.

    Google Scholar 

  20. Zdanovsky, A., Zdanovskaia, M., Ma, D., Wood, K.V., Almond, B., Wood, M.G. (2004) Rapidly degraded reporter fusion proteins. United States Patent US2004/0146987 A1.

    Google Scholar 

  21. Voon, D.C., Subrata, L.S., Baltic, S., Leu, M.P., Whiteway, J.M., Wong, A., Knight, S.A., Christiansen, F.T., Daly, J.M. (2005) Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system. Nucleic Acids Res. 33(3):e27.

    Google Scholar 

  22. Urlaub, G., Kas, E., Carothers, A.M., Chasin, L.A. (1983) Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell. 33:405–412.

    Google Scholar 

Download references

Acknowledgments

The author would like to thank Professor Daniel I.C. Wang and Professor Miranda M.G.S. Yap for their guidance and advice, S.F. Lim for providing the rhIFNγ construct, and support from Bioprocessing Technology Institute (Biomedical Sciences Institutes, A*STAR) and Singapore-MIT Alliance.

Author information

Authors and Affiliations

  1. Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

    Say Kong Ng

Authors
  1. Say Kong Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Say Kong Ng .

Editor information

Editors and Affiliations

  1. SAIC-Frederick, Inc., Protein Expression Laboratory, National Cancer Institute, Frederick, Boyles Street 1050, Frederick, 21702, Maryland, USA

    James L Hartley

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Ng, S.K. (2012). Generation of High-Expressing Cells by Methotrexate Amplification of Destabilized Dihydrofolate Reductase Selection Marker. In: Hartley, J. (eds) Protein Expression in Mammalian Cells. Methods in Molecular Biology, vol 801. Humana Press. https://doi.org/10.1007/978-1-61779-352-3_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-352-3_11

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-351-6

  • Online ISBN: 978-1-61779-352-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation