Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells.