Tabriz University of Medical Sciences Advanced Pharmaceutical Bulletin 2228-5881 16 3 2026 09 30 Comparative Analysis of MAR and Terminator Elements in Controlling Stable EPO Expression in CHO-K1 Cells 562 572 10.34172/apb.45955 EN Fatemeh Jalali https://orcid.org/0009-0007-9895-1435 Shahriyar Abdoli Fatemeh Shamsabadi Ahad Yamchi Anvarsadat Kianmehr Majid Shahbazi https://orcid.org/0000-0002-4143-8282 Journal Article 10.34172/apb.45955 2025 06 25 2026 06 22 Introduction: Chromosome remodeling, mRNA 3’ end extension diversity, mRNA maturation, and the utilization of upstream and downstream DNA elements have been shown to significantly impact the production of recombinant proteins. In this study, we employed the human beta-globin Matrix Attachment Region (β-MAR) as an upstream element and the human beta-globin terminator (β-TERM) as a downstream section to augment erythropoietin (EPO) production. Methods: Four plasmids expressing the EPO transgene, after optimization for codon bias and secondary structure of the EPO mRNA, were transfected into CHO-K1 cells. Two weeks after antibiotic selection, pooled stable clones were evaluated for gene copy number (GCN), mRNA quantification, and EPO expression level. Results: Our results revealed a significant increase in the amount of EPO secreted by stable CHO cells in the presence of β-MAR. At the same time, β-TERM had no noticeable impact on EPO production. As expected, the presence of the β-MAR sequence resulted in a higher gene copy number with 15 gene copy per cell (GCN/cell) and mRNA quantity with 5.3 fold change, leading to a much greater secretion of EPO with 28.3 mIU/ml compared to the β-TERM sequence with 3 GCN/cell, 2.8 mRNA fold change and 8.2 mIU/ml of EPO quantity. In the other construct incorporating both β-MAR and β-TERM elements, a gene copy number (GCN) of 7.0 per cell was detected, accompanied by a 4.58-fold elevation in mRNA expression and a secreted erythropoietin (EPO) concentration of 22.45 mIU/ml. In contrast, the construct devoid of both β-MAR and β-TERM (used as the calibrator) elements demonstrated a GCN of 1.0 per cell, an mRNA expression fold change of 1.0, and an EPO concentration of 8.35 mIU/ml. Conclusion: This experiment further supports previous studies, demonstrating the significant impact of the β-MAR element on recombinant protein production in stable CHO-K1 cells.