Skip to main content

Using Matrix Attachment Regions to Improve Recombinant Protein Production

  • Protocol
  • First Online:
Protein Expression in Mammalian Cells

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

Abstract

Chinese hamster ovary (CHO) cells are the system of choice for the production of complex molecules, such as monoclonal antibodies. Despite significant progress in improving the yield from these cells, the process to the selection, identification, and maintenance of high-producing cell lines remains cumbersome, time consuming, and often of uncertain outcome. Matrix attachment regions (MARs) are DNA sequences that help generate and maintain an open chromatin domain that is favourable to transcription and may also facilitate the integration of several copies of the transgene. By incorporating MARs into expression vectors, an increase in the proportion of high-producer cells as well as an increase in protein production are seen, thereby reducing the number of clones to be screened and time to production by as much as 9 months. In this chapter, we describe how MARs can be used to increase transgene expression and provide protocols for the transfection of CHO cells in suspension and detection of high-producing antibody cell clones.

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

Access this chapter

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

Institutional subscriptions

References

  1. Walsh, G. (2006) Biopharmaceutical benchmarks 2006. Nat Biotechnol, 24, 769–776.

    Article  PubMed  CAS  Google Scholar 

  2. Wurm, F.M. (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol, 22, 1393–1398.

    Article  PubMed  CAS  Google Scholar 

  3. Kaufman, R.J. (1990) Selection and coamplification of heterologous genes in mammalian cells. Methods Enzymol, 185, 537–566.

    Article  PubMed  CAS  Google Scholar 

  4. Kim, N., Byun, T. and Lee, G. (2001) Key determinants in the occurrence of clonal variation in humanized antibody expression of CHO cells during dihydrofolate reductase mediated gene amplification. Biotechnol Prog, 17, 69–75.

    Article  PubMed  Google Scholar 

  5. Kim, S., Kim, N., Ryu, C., Hong, H. and Lee, G. (1998) Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol Bioeng, 58, 73–84.

    Article  PubMed  CAS  Google Scholar 

  6. Chusainow, J., Yang, Y.S., Yeo, J.H., Toh, P.C., Asvadi, P., Wong, N.S. and Yap, M.G. (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng, 102, 1182–1196.

    Article  PubMed  CAS  Google Scholar 

  7. Pilbrough, W., Munro, T.P. and Gray, P. (2009) Intraclonal protein expression heterogeneity in recombinant CHO cells. PLoS One, 4, e8432.

    Article  PubMed  Google Scholar 

  8. Raj, A., Peskin, C., Tranchina, D., Vargas, D. and Tyagi, S. (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol, 4, e309.

    Article  PubMed  Google Scholar 

  9. Gorman, C., Arope, S., Grandjean, M., Girod, P. and Mermod, N. (2009) Use of MAR elements to increase the production of recombinant proteins. Cell Engineering, 6, 1–32.

    Article  CAS  Google Scholar 

  10. Yang, Y., Mariati, Chusainow, J. and Yap, M.G. (2010) DNA methylation contributes to loss in productivity of monoclonal antibody-producing CHO cell lines. J Biotechnol, 147, 180–185.

    Google Scholar 

  11. Ferrai, C., Xie, S.Q., Luraghi, P., Munari, D., Ramirez, F., Branco, M.R., Pombo, A. and Crippa, M.P. (2010) Poised transcription factories prime silent uPA gene prior to activation. PLoS Biol, 8, e1000270.

    Article  PubMed  Google Scholar 

  12. Galbete, J.L., Buceta, M. and Mermod, N. (2009) MAR elements regulate the probability of epigenetic switching between active and inactive gene expression. Mol Biosyst, 5, 143–150.

    Article  PubMed  Google Scholar 

  13. Kwaks, T.H. and Otte, A.P. (2006) Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells. Trends Biotechnol, 24, 137–142.

    Article  PubMed  CAS  Google Scholar 

  14. Girod, P.A., Zahn-Zabal, M. and Mermod, N. (2005) Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines. Biotechnol Bioeng, 91, 1–11.

    Article  PubMed  CAS  Google Scholar 

  15. Zahn-Zabal, M., Kobr, M., Girod, P.A., Imhof, M., Chatellard, P., de Jesus, M., Wurm, F. and Mermod, N. (2001) Development of stable cell lines for production or regulated expression using matrix attachment regions. J Biotechnol, 87, 29–42.

    Article  PubMed  CAS  Google Scholar 

  16. Phi-Van, L., von Kries, J.P., Ostertag, W. and Stratling, W.H. (1990) The chicken lysozyme 5′ matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol, 10, 2302–2307.

    PubMed  CAS  Google Scholar 

  17. Girod, P.A., Nguyen, D.Q., Calabrese, D., Puttini, S., Grandjean, M., Martinet, D., Regamey, A., Saugy, D., Beckmann, J.S., Bucher, P. et al. (2007) Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nat Methods, 4, 747–753.

    Article  PubMed  CAS  Google Scholar 

  18. Dang, Q., Auten, J. and Plavec, I. (2000) Human beta interferon scaffold attachment region inhibits de novo methylation and confers long-term, copy number-dependent expression to a retroviral vector. J Virol, 74, 2671–2678.

    Article  PubMed  CAS  Google Scholar 

  19. Liebich, I., Bode, J., Frisch, M. and Wingender, E. (2002) S/MARt DB: a database on scaffold/matrix attached regions. Nucleic Acids Res, 30, 372–374.

    Article  PubMed  CAS  Google Scholar 

  20. Harraghy, N., Gaussin, A. and Mermod, N. (2008) Sustained transgene expression using MAR elements. Curr Gene Ther, 8, 353–366.

    Article  PubMed  CAS  Google Scholar 

  21. Kim, J.-M., Kim, J.-S., Park, D.-H., Kang, H., Yoon, J., Baek, K. and Yoon, Y. (2004) Improved recombinant gene expression in CHO cells using matrix attachment regions. J Biotechnol, 107, 95–105.

    Article  PubMed  CAS  Google Scholar 

  22. Kim, J., Yoon, Y., Hwang, H.-Y., Park, J., Yu, S., Lee, J., Baek, K. and Yoon, J. (2005) Efficient selection of stable Chinese hamster ovary (CHO) cell lines for expression of recombinant proteins by using human interferon b SAR element. Biotechnol Prog, 21, 933–937.

    Article  PubMed  CAS  Google Scholar 

  23. Varghese, J., Alves, W., Brill, B., Wallace, M., Calabrese, D., Regamey, A. and Girod, P. (2008) Rapid development of high-performance, stable mammalian cell lines for improved clinical development. Bioprocess J, 7, 30–36.

    CAS  Google Scholar 

  24. Evans, K., Ott, S., Hansen, A., Koentges, G. and Wernisch, L. (2007) A comparative study of S/MAR prediction tools. BMC Bioinformatics, 8, 71.

    Article  PubMed  Google Scholar 

  25. Singh, G.B., Kramer, J.A. and Krawetz, S.A. (1997) Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucleic Acids Res, 25, 1419–1425.

    Article  PubMed  CAS  Google Scholar 

  26. Frisch, M., Frech, K., Klingenhoff, A., Cartharius, K., Liebich, I. and Werner, T. (2002) In silico prediction of scaffold/matrix attachment regions in large genomic sequences. Genome Res, 12, 349–354.

    Article  PubMed  CAS  Google Scholar 

  27. Jenke, A.C., Stehle, I.M., Herrmann, F., Eisenberger, T., Baiker, A., Bode, J., Fackelmayer, F.O. and Lipps, H.J. (2004) Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome. Proc Natl Acad Sci USA, 101, 11322–11327.

    Article  PubMed  CAS  Google Scholar 

  28. Piechaczek, C., Fetzer, C., Baiker, A., Bode, J. and Lipps, H.J. (1999) A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucleic Acids Res, 27, 426–428.

    Article  PubMed  CAS  Google Scholar 

  29. Stehle, I.M., Postberg, J., Rupprecht, S., Cremer, T., Jackson, D.A. and Lipps, H.J. (2007) Establishment and mitotic stability of an extra-chromosomal mammalian replicon. BMC Cell Biol, 8, 33.

    Article  PubMed  Google Scholar 

  30. Giannakopoulos, A., Stavrou, E.F., Zarkadis, I., Zoumbos, N., Thrasher, A.J. and Athanassiadou, A. (2009) The functional role of S/MARs in episomal vectors as defined by the stress-induced destabilization profile of the vector sequences. J Mol Biol, 387, 1239–1249.

    Article  PubMed  CAS  Google Scholar 

  31. Rosser, M.P., Xia, W., Hartsell, S., McCaman, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P. and Cobb, R.R. (2005) Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expr Purif, 40, 237–243.

    Article  PubMed  CAS  Google Scholar 

  32. Albano, C.R., Randers-Eichhorn, L., Bentley, W.E. and Rao, G. (1998) Green fluorescent protein as a real time quantitative reporter of heterologous protein production. Biotechnol Prog, 14, 351–354.

    Article  PubMed  CAS  Google Scholar 

  33. Meng, Y.G., Liang, J., Wong, W.L. and Chisholm, V. (2000) Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene, 242, 201–207.

    Article  PubMed  CAS  Google Scholar 

  34. Pick, H.M., Meissner, P., Preuss, A.K., Tromba, P., Vogel, H. and Wurm, F.M. (2002) Balancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human Rhesus-D IgG1 synthesis. Biotechnol Bioeng, 79, 595–601.

    Article  PubMed  CAS  Google Scholar 

  35. Brezinsky, S.C., Chiang, G.G., Szilvasi, A., Mohan, S., Shapiro, R.I., MacLean, A., Sisk, W. and Thill, G. (2003) A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J Immunol Methods, 277, 141–155.

    Article  PubMed  CAS  Google Scholar 

  36. Bergman, L.W., Harris, E. and Kuehl, W.M. (1981) Glycosylation causes an apparent block in translation of immunoglobulin heavy chain. J Biol Chem, 256, 701–706.

    PubMed  CAS  Google Scholar 

  37. Bibila, T. and Flickinger, M.C. (1991) A structured model for monoclonal antibody synthesis in exponentially growing and stationary phase hybridoma cells. Biotechnol Bioeng, 37, 210–226.

    Article  PubMed  CAS  Google Scholar 

  38. Schlatter, S., Stansfield, S.H., Dinnis, D.M., Racher, A.J., Birch, J.R. and James, D.C. (2005) On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells. Biotechnol Prog, 21, 122–133.

    Article  PubMed  CAS  Google Scholar 

  39. English, C., Merson, S. and Keer, J. (2006) Use of elemental analysis to determine comparative performance of established DNA quantification methods. Anal Chem, 78, 4630–4633.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Nicolas Mermod .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Harraghy, N., Buceta, M., Regamey, A., Girod, PA., Mermod, N. (2012). Using Matrix Attachment Regions to Improve Recombinant Protein Production. 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_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-352-3_7

  • 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