Two human MARs effectively increase transgene expression in transfected CHO cells

Abstract

Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR‐3 and MAR‐7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real‐time PCR. The results showed that the expression level of eGFP of cells transfected with MAR‐containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR‐7 was higher than that of MAR‐3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR‐3 and MAR‐7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.

Novel short synthetic matrix attachment region for enhancing transgenic expression in recombinant Chinese hamster ovary cells

“…We previously screened a series of MAR sequences and designed synthetic MARs based on the characteristics of MAR sequences to improve the levels and stability of transgenic expression in CHO cells . We also analyzed the molecular characteristics of MAR elements that are requisite in improving transgenic expression in CHO cells .…”

“…We previously screened a series of MAR sequences and designed synthetic MARs based on the characteristics of MAR sequences to improve the levels and stability of transgenic expression in CHO cells. 14,19,25,26 We also analyzed the molecular characteristics of MAR elements that are requisite in improving transgenic expression in CHO cells. 21 Bioinformatic analysis showed that the elements encode a MAR-like motif and some transcription factor binding sites such as CEBP, CTCF, GSH, and SATB1binding motifs may be conducive to the MAR-mediated augmentation of transgenic expression.…”

Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells

“…An appropriate combination of expression vector elements can improve the expression of recombinant proteins, overcome gene silencing, and increase the stability of transgene expression. At present, there have been reports on matrix attachment regions (MARs) ( Gao et al, 2019 ; Jia et al, 2019 ; Li et al, 2019 ), ubiquitous chromatin opening elements ( Saunders et al, 2015 ), cis-acting factors ( Wang et al, 2018 ), stabilizing Anti Repressor elements ( Hoeksema et al, 2011 ), introns ( Osabe et al, 2017 ), internal ribosome entry sites (IRES) ( Chai et al, 2018 ), exons ( Lu et al, 2017 ) and promoters ( Wang et al, 2017 ; Yi et al, 2020 ), and found that these elements can improve the expression of recombinant proteins to some extent.…”

Advances in the Development and the Applications of Nonviral, Episomal Vectors for Gene Therapy

“…Interestingly, the same group reported that for mouse liver 65 or human mesenchymal stem cells, 52 both the vector and the transgene should be CpG free, whereas in mouse skeletal muscle, 65 removing the CpG motifs from the vector alone was sufficient to maintaining expression for about 100 days relative to control vector. Noticeably, other vectors with similar dual S/MAR regions (IFNβ S/MAR and an immunoglobulin S/MAR 66 or short chimeric MARs 67 ) promote higher colony formation, transgene expression levels, and maintenance in CHO cells relative to one S/MAR, suggesting that more than one S/MAR may augment the vector's activity and function further.…”