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.
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References
Walsh, G. (2006) Biopharmaceutical benchmarks 2006. Nat Biotechnol, 24, 769–776.
Wurm, F.M. (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol, 22, 1393–1398.
Kaufman, R.J. (1990) Selection and coamplification of heterologous genes in mammalian cells. Methods Enzymol, 185, 537–566.
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.
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.
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.
Pilbrough, W., Munro, T.P. and Gray, P. (2009) Intraclonal protein expression heterogeneity in recombinant CHO cells. PLoS One, 4, e8432.
Raj, A., Peskin, C., Tranchina, D., Vargas, D. and Tyagi, S. (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol, 4, e309.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Harraghy, N., Gaussin, A. and Mermod, N. (2008) Sustained transgene expression using MAR elements. Curr Gene Ther, 8, 353–366.
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.
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.
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.
Evans, K., Ott, S., Hansen, A., Koentges, G. and Wernisch, L. (2007) A comparative study of S/MAR prediction tools. BMC Bioinformatics, 8, 71.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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
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DOI: https://doi.org/10.1007/978-1-61779-352-3_7
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