Abstract
The gradual loss of recombinant protein expression in CHO cell lines during prolonged subculture is a common issue, referred to as instability, which seriously affects the industrial production processes of therapeutic proteins. Loss of recombinant gene copies, due to the genetic instability of CHO cells, and epigenetic silencing of transgene sequences, are the main reported causes of production instability. To increase our understanding on the molecular mechanisms inherent to CHO cells involved in production instability, we explored the molecular features of stable and unstable antibody producing cell lines obtained without gene amplification, to exclude the genetic instability induced by the gene amplification process. The instability of recombinant antibody production during long-term culture was caused by a 48–53 % decrease in recombinant mRNA levels without significant loss of recombinant gene copies, but accompanied by a ~45 % decrease in histone H3 acetylation (H3ac). Thus, our results suggest a critical role of H3ac in the stability of recombinant protein production.
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References
Bailey LA, Hatton D, Field R, Dickson AJ (2012) Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture. Biotechnol Bioeng 109(8):2093–2103
Berger SL (2007) The complex language of chromatin regulation during transcription. Nature 447:407–412
Choi KH, Basma H, Singh J, Cheng PW (2005) Activation of CMV promoter-controlled glycosyltransferase and beta-galactosidase glycogenes by butyrate, tricostatin A, and 5-aza-2′-deoxycytidine. Glycoconj J 22(1–2):63–69
Chusainow J, Yang YS, Yeo JH, Toh PC, Asvadi P, Wong NS, Yap MG (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102(4):1182–1196
Ciudad CJ, Urlaub G, Chasin LA (1988) Deletion analysis of the Chinese hamster dihydrofolate reductase gene promoter. J Biol Chem 263:16274–16282
Kaufman RJ, Sharp PA, Latt SA (1983) Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences. Mol Cell Biol 3(4):699–711
Kaufman WL, Kocman I, Agrawal V, Rahn HP, Besser D, Gossen M (2008) Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation. Nucleic Acids Res 36(17):e111
Kim JM, Kim JS, Park DH, Kang HS, Yoon J, Baek K, Yoon Y (2004) Improved recombinant gene expression in CHO cells using matrix attachment regions. J Biotechnol 107:95–105
Kim M, O’Callaghan PM, Droms KA, James DC (2011) A Mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnol Bioeng 108:2434–2446
Lattenmayer C, Loeschel M, Steinfellner W, Trummer E, Mueller D, Schriebl K, Vorauer-Uhl K, Katinger H, Kunert R (2006) Identification of transgene integration loci of different highly expressing recombinant CHO cell lines by FISH. Cytotechnology 51(3):171–182
Mutskov V, Felsenfeld G (2004) Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9. EMBO J 23(1):138–149
Riu E, Chen ZY, Xu H, He CY, Kay MA (2007) Histone modifications are associated with the persistence or silencing of vector-mediated transgene expression in vivo. Mol Ther 15(7):1348–1355
Shahbazian MD, Grunstein M (2007) Functions of site-specific histone acetylation and deacetylation. Annu Rev Biochem 76:75–100
Yan C, Boyd DD (2006) Histone H3 Acetylation and H3 K4 methylation define distinct chromatin regions permissive for transgene expression. Mol Cell Biol 26(17):6357–6371
Yang Y, Mariati, Chusainow J, Yap MG (2010) DNA methylation contributes to loss in productivity of monoclonal antibody-producing CHO cell lines. J Biotechnol 147:180–185
Yin Z, Kong QR, Zhao ZP, Wu ML, Mu YS, Hu K, Liu ZH (2012) Position effect variegation and epigenetic modification of a transgene in a pig model. Genet Mol Res 11(1):355–369
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This work was supported by the R&D Program (10035201) of MKE/KEIT, Korea.
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Paredes, V., Park, J.S., Jeong, Y. et al. Unstable expression of recombinant antibody during long-term culture of CHO cells is accompanied by histone H3 hypoacetylation. Biotechnol Lett 35, 987–993 (2013). https://doi.org/10.1007/s10529-013-1168-8
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DOI: https://doi.org/10.1007/s10529-013-1168-8