3 Biotech

, 9:435 | Cite as

Enhanced transgene expression using two β-globin MARs flanking expression cassettes in stably transfected CHO-K1 cells

  • Jihong Zhang
  • Junhe ZhangEmail author
  • Shan Cheng
  • Wenwen Yang
  • Shijiang Li
Original Article


In this study, we systemically investigated the positions and orientations of matrix attachment regions (MARs) in expression vectors to fully explore the mechanism for improving transgene expression. We constructed 14 vectors that incorporated human β-globin MARs into pIRES-eGFP backbone vectors. The MARs flanked the eGFP expression cassette or promoter in a forward/reverse orientation. After stable transfection into CHO-K1 cells with these vectors, eGFP expression levels were increased significantly relative to that of the control vector (MAR-devoid) when two MARs flanking the expression cassette were incorporated, followed by those at the 5′ site (upstream of the promoter). Simultaneously, the percentage of the eGFP-expressing cells was elevated to some extent. The vector with both MARs in forward orientation flanking the expression cassette yielded the highest transgene expression levels (2.5-fold). The orientation (forward or reverse) of the MARs did not present a significant difference when added in the same site. In addition, transgene expression levels were not exclusively dependent on transgene copy numbers. Bioinformatic analysis indicated that some specific transcription factors may contribute to the transcriptional process. In conclusion, two MARs in a forward orientation and flanking the expression cassette comprised the optimal construct for improving the stable transgene expression in the CHO-K1 cells. The effects may be related to specific transcription factors, such as PRDM1 and REL.


Matrix attachment region Position effect Transgene expression Transcription factor-binding site 



This work was supported by the Grant from the National Natural Science Foundation of China (no. U1604193).

Author contributions

JZ designed, analyzed the experiments and wrote the manuscript. JZ performed the experiments and co-wrote the manuscript. SC and WY analyzed some experiment data and co-wrote the manuscript. SL performed the experiment for vector construction and cultured cells.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.


  1. Arope S, Harraghy N, Pjanic M, Mermod N (2013) Molecular characterization of a human matrix attachment region epigenetic regulator. PLoS One 8:e79262CrossRefGoogle Scholar
  2. Bode J, Benham C, Knopp A, Mielke C (2000) Transcriptional augmentation: modulation of gene expression by scaffold/matrix-attached regions (S/MAR elements). Crit Rev Eukaryot Gene Expr 10:73–90CrossRefGoogle Scholar
  3. Buceta M, Galbete JL, Kostic C, Arsenijevic Y, Mermod N (2011) Use of human MAR elements to improve retroviral vector production. Gene Ther 18:7–13CrossRefGoogle Scholar
  4. Dolgova AS, Dolgov SV (2019) Matrix attachment regions as a tool to influence plant transgene expression. 3 Biotech 9:176CrossRefGoogle Scholar
  5. Galbete JL, Buceta M, Mermod N (2009) MAR elements regulate the probability of epigenetic switching between active and inactive gene expression. Mol Biosyst 5:143–150CrossRefGoogle Scholar
  6. Gaussin A, Modlich U, Bauche C, Niederländer NJ, Schambach A, Duros C, Artus A, Baum C, Cohen-Haguenauer O, Mermod N (2012) CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors. Gene Ther 19:15–24CrossRefGoogle Scholar
  7. Girod PA, Mermod N (2003) Use of scaffold/matrix attachment regions for protein production. Gene Transf Expr Mamm Cells 38:359–379CrossRefGoogle Scholar
  8. Girod PA, Zahn-Zabal M, Mermod N (2005) Use of the chicken lysozyme matrix attachment region to generate high producer CHO cell lines. Biotechnol Bioeng 91:1–11CrossRefGoogle Scholar
  9. Girod PA, Nguyen DQ, Calabrese D, Puttini S, Grandjean M, Martinet D, Regamey A, Saugy D, Beckmann JS, Bucher P, Mermod N (2007) Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nat Methods 4:747–753CrossRefGoogle Scholar
  10. Grandjean M, Girod PA, Calabrese D, Kostyrko K, Wicht M, Yerly F, Mazza C, Beckmann JS, Martinet D, Mermod N (2011) High-level transgene expression by homologous recombination-mediated gene transfer. Nucleic Acids Res 39:e104CrossRefGoogle Scholar
  11. Heng HH, Goetze S, Ye CJ, Liu G, Stevens JB, Bremer SW, Wykes SM, Bode J, Krawetz SA (2004) Chromatin loops are selectively anchored using scaffold/matrix-attachment regions. J Cell Sci 117:999–1008CrossRefGoogle Scholar
  12. Jia YL, Guo X, Ni TJ, Lu JT, Wang XY, Wang TY (2019a) Novel short synthetic matrix attachment region for enhancing transgenic expression in recombinant Chinese hamster ovary cells. J Cell Biochem 120:18478–18486PubMedGoogle Scholar
  13. Jia YL, Guo X, Wang XC, Wang TY (2019b) Human genome-derived TOP1 matrix attachment region enhances transgene expression in the transfected CHO cells. Biotechnol Lett 41:701–709CrossRefGoogle Scholar
  14. 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 107:95–105CrossRefGoogle Scholar
  15. Kwaks TH, Otte AP (2006) Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells. Trends Biotechnol 24:137–142CrossRefGoogle Scholar
  16. Li Q, Dong W, Wang T, Liu Z, Wang F, Wang X, Zhao C, Zhang J, Wang L (2013) Effect of β-globin MAR characteristic elements on transgene expression. Mol Med Rep 7:1871–1874CrossRefGoogle Scholar
  17. Li Q, Zhao CP, Lin Y, Song C, Wang F, Wang TY (2019) Two human MARs effectively increase transgene expression in transfected CHO cells. J Cell Mol Med 23:1613–1616CrossRefGoogle Scholar
  18. Linnemann AK, Krawetz SA (2009) Silencing by nuclear matrix attachment distinguishes cell-type specificity: association with increased proliferation capacity. Nucleic Acids Res 37:2779–2788CrossRefGoogle Scholar
  19. Linnemann AK, Platts AE, Krawetz SA (2009) Differential nuclear scaffold/matrix attachment marks expressed genes. Hum Mol Genet 18:645–654CrossRefGoogle Scholar
  20. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408CrossRefGoogle Scholar
  21. Majocchi S, Aritonovska E, Mermod N (2014) Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes. Nucleic Acids Res 42:193–204CrossRefGoogle Scholar
  22. Mathelier A, Fornes O, Arenillas DJ, Chen CY, Denay G, Lee J, Shi W, Shyr C, Tan G, Worsley-Hunt R, Zhang AW, Parcy F, Lenhard B, Sandelin A, Wasserman WW (2016) JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles. Nucleic Acids Res 44:D110–D115CrossRefGoogle Scholar
  23. Sjeklocha L, Chen Y, Daly MC, Steer CJ, Kren BT (2011) β-globin matrix attachment region improves stable genomic expression of the sleeping beauty transposon. J Cell Biochem 112:2361–2375CrossRefGoogle Scholar
  24. Tian ZW, Xu DH, Wang TY, Wang XY, Xu HY, Zhao CP, Xu GH (2018) Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells. J Cell Mol Med 22:1095–1102PubMedGoogle Scholar
  25. Varghese J, Alves W, Brill B, Wallace M, Calabrese D, Regamey A, Girod P (2008) Rapid development of high-performance, stable mammalian cell lines for improved clinical development. Bioprocess J 7:30–36CrossRefGoogle Scholar
  26. Wang TY, Yang R, Qin C, Wang L, Yang XJ (2008) Enhanced expression of transgene in CHO cells using matrix attachment region. Cell Biol Int 32:1279–1283CrossRefGoogle Scholar
  27. Wang TY, Zhang JH, Jing CQ, Yang XJ, Lin JT (2010) Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells. Cell Biol Int 34:141–145CrossRefGoogle Scholar
  28. Wang F, Wang TY, Tang YY, Zhang JH (2012) Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells. Gene 500:59–62CrossRefGoogle Scholar
  29. Wang XJ, Wang J, Wang YY, Guo YJ, Chu BB, Yang GY (2016) Sus scrofa matrix attachment region enhances expression of the PiggyBac system transfected into HEK293T cells. Biotechnol Lett 38:949–958CrossRefGoogle Scholar
  30. Wang W, Guo X, Li YM, Wang XY, Yang XJ, Wang YF, Wang TY (2018) Enhanced transgene expression using cis-acting elements combined with the EF1 promoter in a mammalian expression system. Eur J Pharm Sci 123:539–545CrossRefGoogle Scholar
  31. Yu J, Bock JH, Slightom JL, Villeponteau B (1994) A 5′ β-globin matrix attachment region and the polyoma enhancer together confer position-independent transcription. Gene 139:139–145CrossRefGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.School of Basic Medical SciencesXinxiang Medical UniversityXinxiangChina
  2. 2.International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of HenanXinxiangChina
  3. 3.The First Affiliated Hospital of Xinxiang Medical UniversityWeihuiChina

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