Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells

Abstract

Low-level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR-6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR-6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP-B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP-B, DHFR intron MAR element and MAR-6. Additionally, as expected, the three MAR-containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non-MAR-containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP-B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.

Two human MARs effectively increase transgene expression in transfected CHO cells

“…In the present study, MAR‐3 and MAR‐7 enhanced the transgene expression levels in stably transfected CHO cells, which are consistent with the previous studies . In addition, MAR‐7 showed the higher enhancing activity compared with MAR‐3.…”

“…In the present study, MAR-3 and MAR-7 enhanced the transgene expression levels in stably transfected CHO cells, which are consistent with the previous studies. [8][9][10] In addition, MAR-7 showed the higher enhancing activity compared with MAR-3. We suspect that the backbone, motif composition of MARs may determine their effect on transgene expression.…”

“…A pIRES-EGFP plasmid vector, as described in the previous study, [9][10][11] was used in the present study.…”

“…Therefore, MARs are widely used to improve the high level of expression of transgenic genes and to prevent epigenetic silencing by blocking the transmission of heterochromatin . Up to now, it has been reported that several MAR elements can increase the level of transgene expression as well as the proportion of positive colonies in CHO cell expression systems, and also reduce the differences of expression between different transformed strains …”

Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells

“…The expression vector pIRES-Neo was purchased from Clontech company (Clontech, Mountain View, USA). The pIRES-EGFP vector was constructed by sub-cloning EGFP gene from pEGFP-C1 (Clontech, Mountain View, USA) to the pIRES-neo vector 30 . Ten IRES fragments including immunoglobulin heavy chain binding protein (BIP), cationic amino acid transporter1 (CAT-1), c-myc proto-oncogene (c-myc), Hepatitis C virus (HCV), vascular endothelial growth factor (VEGF) and type1 collagen inducible protein (VCIP), Apoptotic protease activating factor 1 (Apaf-1), Encephalomyocarditis virus (EMCV), Human rhinovirus (HRV) and NF-kappa B repressing factor (NRF) (Table 1 ) were synthesized by the company (sequence information was seen in Supplement material 1 ) and inserted between EGFP and Neo of the pIRES-EGFP vector to generate ten fusing vectors (pIRES-EGFP1-10), respectively.…”