, Volume 69, Issue 3, pp 451–460 | Cite as

Identification of regulatory motifs in the CHO genome for stable monoclonal antibody production

  • Yasuhiro Takagi
  • Tomomi Yamazaki
  • Kenji Masuda
  • Shigeaki Nishii
  • Bunsei Kawakami
  • Takeshi OmasaEmail author
Original Research


Chinese hamster ovary (CHO) cell lines are widely used for therapeutic protein production. When a transgene is integrated into the genome of a CHO cell, the expression level is highly dependent on the site of integration because of positional effects such as gene silencing. To overcome negative positional effects and establish stable CHO cell lines with high productivity, several regulatory DNA elements are used in vector construction. Previously, we established the CHO DR1000L-4N cell line, a stable and high copy number Dhfr gene-amplified cell line. It was hypothesized that the chromosomal location of the exogenous gene-amplified region in the CHO DR1000L-4N genome contains regulatory motifs for stable protein production. Therefore, we isolated DNA regulatory motifs from the CHO DR1000L-4N cell line and determined whether these motifs act as an insulator. Our results suggest that stable expression of a transgene can be promoted by the CHO genome sequence, and it would be a powerful tool for therapeutic protein manufacturing.


Chinese hamster ovary cells Therapeutic antibody Insulator Epigenetics 



This work was partly patented as Japan patent 4568378 and partly supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. Authors also appreciate to Mr. Shuichi Kimura for detail discussion.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Bao L, Zhou M, Cui Y (2008) CTCFBSDB: a CTCF-binding site database for characterization of vertebrate genomic insulators. Nucleic Acids Res 36:D83–D87. doi: 10.1093/nar/gkm875 CrossRefGoogle Scholar
  2. Bell AC, West AG, Felsenfeld G (2001) Insulators and boundaries: versatile regulatory elements in the eukaryotic genome. Science 291:447–450. doi: 10.1126/science.291.5503.447 CrossRefGoogle Scholar
  3. Blasco MA (2007) The epigenetic regulation of mammalian telomeres. Nat Rev Genet 8:299–309. doi: 10.1038/nrg2047 CrossRefGoogle Scholar
  4. Ewing B, Green P (1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8:186–194. doi: 10.1101/gr.8.3.186 CrossRefGoogle Scholar
  5. Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185. doi: 10.1101/gr.8.3.175 CrossRefGoogle Scholar
  6. Gonzalo S, Jaco I, Fraga MF, Chen T, Li E, Esteller M, Blasco MA (2006) DNA methyltransferases control telomere length and telomere recombination in mammalian cells. Nat Cell Biol 8:416–424. doi: 10.1038/ncb1386 CrossRefGoogle Scholar
  7. Gordon D, Abajian C, Green P (1998) Consed: a graphical tool for sequence finishing. Genome Res 8:195–202. doi: 10.1101/gr.8.3.195 CrossRefGoogle Scholar
  8. Herold M, Bartkuhn M, Renkawitz R (2012) CTCF: insights into insulator function during development. Development 139:1045–1057. doi: 10.1242/dev.065268 CrossRefGoogle Scholar
  9. Izumi M, Gilbert DM (1999) Homogeneous tetracycline-regulatable gene expression in mammalian fibroblasts. J Cell Biochem 76:280–289CrossRefGoogle Scholar
  10. Kang SY, Kim YG, Kang S, Lee HW, Lee EG (2016) A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells. Biotechnol J 11:633–641. doi: 10.1002/biot.201500464 CrossRefGoogle Scholar
  11. Kim HY (2007) Use of DNA insulator elements and scaffold/matirix-attached regions for enhanced recombinant protein expression. In: Butler M (ed) Cell culture and upstream processing. Taylor & Francis, New York, pp 19–36Google Scholar
  12. 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. doi: 10.1016/j.jbiotec.2003.09.015 CrossRefGoogle Scholar
  13. Kim TH, Abdullaev ZK, Smith AD, Ching KA, Loukinov DI, Green RD, Zhang MQ, Lobanenkov VV, Ren B (2007) Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell 128:1231–1245. doi: 10.1016/j.cell.2006.12.048 CrossRefGoogle Scholar
  14. 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. doi: 10.1002/bit.23189 CrossRefGoogle Scholar
  15. Koo TY, Park JH, Park HH, Park TH (2009) Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-β in serum-free suspension culture of CHO cells. Process Biochem 44:146–153. doi: 10.1016/j.procbio.2008.09.018 CrossRefGoogle Scholar
  16. Kwaks TH, Otte AP (2006) Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells. Trends Biotechnol 24:137–142. doi: 10.1016/j.tibtech.2006.01.007 CrossRefGoogle Scholar
  17. Li J, Sun X, Zhang Y (2005) Improvement of hepatitis B surface antigen expression by dimethyl sulfoxide in the culture of recombinant Chinese hamster ovary cells. Process Biochem 41:317–322. doi: 10.1016/j.procbio.2005.08.017 CrossRefGoogle Scholar
  18. Majocchi S, Aritonovska E, Mermod N (2014) Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes. Nucleic Acids Res 42:193–204. doi: 10.1093/nar/gkt880 CrossRefGoogle Scholar
  19. Maksimenko O, Gasanov NB, Georgiev P (2015) Regulatory elements in vectors for efficient generation of cell lines producing target proteins. Acta Naturae 7:15–26Google Scholar
  20. Moon H, Filippova G, Loukinov D, Pugacheva E, Chen Q, Smith ST, Munhall A, Grewe B, Bartkuhn M, Arnold R, Burke LJ, Renkawitz-Pohl R, Ohlsson R, Zhou J, Renkawitz R, Lobanenkov V (2005) CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator. EMBO Rep 6:165–170. doi: 10.1038/sj.embor.7400334 CrossRefGoogle Scholar
  21. Omasa T (2002) Gene amplification and its application in cell and tissue engineering. J Biosci Bioeng 94:600–605. doi: 10.1016/S1389-1723(02)80201-8 CrossRefGoogle Scholar
  22. Omasa T, Takami T, Ohya T, Kiyama E, Hayashi T, Nishii H, Miki H, Kobayashi K, Honda K, Ohtake H (2008) Overexpression of GADD34 enhances production of recombinant human antithrombin III in Chinese hamster ovary cells. J Biosci Bioeng 106:568–573. doi: 10.1263/Jbb.106.568 CrossRefGoogle Scholar
  23. Omasa T, Cao Y, Park JY, Takagi Y, Kimura S, Yano H, Honda K, Asakawa S, Shimizu N, Ohtake H (2009) Bacterial artificial chromosome library for genome-wide analysis of Chinese hamster ovary cells. Biotechnol Bioeng 104:986–994. doi: 10.1002/bit.22463 CrossRefGoogle Scholar
  24. Ono K, Kamihira M, Kuga Y, Matsumoto H, Hotta A, Itoh T, Nishijima K, Nakamura N, Matsuda H, Iijima S (2003) Production of anti-prion scFv-Fc fusion proteins by recombinant animal cells. J Biosci Bioeng 95:231–238. doi: 10.1016/S1389-1723(03)80022-1 CrossRefGoogle Scholar
  25. Palazzoli F, Bire S, Bigot Y, Bonnin-Rouleux F (2011) Landscape of chromatin control element patents: positioning effects in pharmaceutical bioproduction. Nat Biotechnol 29:593–597. doi: 10.1038/nbt.1907 CrossRefGoogle Scholar
  26. Park JY, Takagi Y, Yamatani M, Honda K, Asakawa S, Shimizu N, Omasa T, Ohtake H (2010) Identification and analysis of specific chromosomal region adjacent to exogenous Dhfr-amplified region in Chinese hamster ovary cell genome. J Biosci Bioeng 109:504–511. doi: 10.1016/j.jbiosc.2009.10.019 CrossRefGoogle Scholar
  27. Perrod S, Gasser SM (2003) Long-range silencing and position effects at telomeres and centromeres: parallels and differences. Cell Mol Life Sci 60:2303–2318. doi: 10.1007/s00018-003-3246-x CrossRefGoogle Scholar
  28. Pikaart MJ, Recillas-Targa F, Felsenfeld G (1998) Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev 12:2852–2862. doi: 10.1101/gad.12.18.2852 CrossRefGoogle Scholar
  29. Reichert JM (2015) Antibodies to watch in 2015. MAbs 7:1–8. doi: 10.4161/19420862.2015.988944 CrossRefGoogle Scholar
  30. Rincon-Arano H, Furlan-Magaril M, Recillas-Targa F (2007) Protection against telomeric position effects by the chicken cHS4 beta-globin insulator. Proc Natl Acad Sci USA 104:14044–14049. doi: 10.1073/pnas.0704999104 CrossRefGoogle Scholar
  31. Saunders F, Sweeney B, Antoniou MN, Stephens P, Cain K (2015) Chromatin function modifying elements in an industrial antibody production platform–comparison of UCOE, MAR, STAR and cHS4 elements. PLoS ONE 10:e0120096. doi: 10.1371/journal.pone.0120096 CrossRefGoogle Scholar
  32. Scott KC, Merrett SL, Willard HF (2006) A heterochromatin barrier partitions the fission yeast centromere into discrete chromatin domains. Curr Biol 16:119–129. doi: 10.1016/j.cub.2005.11.065 CrossRefGoogle Scholar
  33. Wakimoto BT (1998) Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila. Cell 93:321–324. doi: 10.1016/S0092-8674(00)81159-9 CrossRefGoogle Scholar
  34. West AG, Gaszner M, Felsenfeld G (2002) Insulators: many functions, many mechanisms. Genes Dev 16:271–288. doi: 10.1101/gad.954702 CrossRefGoogle Scholar
  35. Xie X, Mikkelsen TS, Gnirke A, Lindblad-Toh K, Kellis M, Lander ES (2007) Systematic discovery of regulatory motifs in conserved regions of the human genome, including thousands of CTCF insulator sites. Proc Natl Acad Sci USA 104:7145–7150. doi: 10.1073/pnas.0701811104 CrossRefGoogle Scholar
  36. Yoshikawa T, Nakanishi F, Itami S, Kameoka D, Omasa T, Katakura Y, Kishimoto M, Suga K (2000a) Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes. Cytotechnology 33:37–46. doi: 10.1023/A:1008111328771 CrossRefGoogle Scholar
  37. Yoshikawa T, Nakanishi F, Ogura Y, Oi D, Omasa T, Katakura Y, Kishimoto M, Suga K (2000b) Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes. Biotechnol Prog 16:710–715. doi: 10.1021/bp000114e CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Institute of Bioscience and BioindustryTokushima UniversityTokushimaJapan
  2. 2.Graduate School of EngineeringOsaka UniversitySuitaJapan
  3. 3.TOYOBO Co.Ltd.TsurugaJapan
  4. 4.Biotechnology LaboratoriesAstellas Pharma Inc.Tsukuba-shiJapan

Personalised recommendations