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.


| INTRODUCTION
Mammalian cell expression systems are generally the preferred platform for producing the recombinant therapy proteins, as they can generate the large and complex proteins with post-translational modifications similar to those produced in humans. 1,2 More than 70% of the recombinant therapeutic proteins were produced by the Chinese hamster ovaries (CHO) cells. 3 Low expression level and transgene silencing of recombinant protein in CHO cells are common problems in the production of recombinant protein. 4,5 Matrix attachment regions (MARs) are the most commonly used epigenetic regulatory element that enhance the integration of plasmids, participate in the formation of chromatin boundaries and enhance transcription. 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. 6,7 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. 8 Although it has been demonstrated that several MARs can increase the transgene expression in transfected CHO cells; however, improvements in function and analyses of the underlying mechanism are necessary. In this study, we identified two new, more powerful MAR elements from the human genome DNA, which can be used to improve the transgene expression in transfected CHO cells.

| Vector construction
A pIRES-EGFP plasmid vector, as described in the previous study, [9][10][11] was used in the present study. Human MAR-3 (GenBank no.

| Cell culture and transfection
Chinese hamster ovaries cells (#A11557-01; Life Technologies, Carlsbad, CA, USA) were cultured in Dulbecco's Modified Eagle's Med-ium+F12 (Gibco, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS; Gibco) and 1% penicillin and streptomycin (Beyotime, Shanghai, China) at 37°C in a humidified incubator with 5% CO 2 . Cells at the exponential growth phase were collected and seeded into 24-well plates at 2 × 10 6 cells/well. The next day, the cells were divided into two groups for transfection with pIRES-MAR-3 and pIRES-MAR-7. pIRES-EGFP was used as a control vector.

| Screening of stably transfected CHO cells
About 48 hours after transfection, the cells were cultured in the presence of 800 μg/mL G418 (Invitrogen) for 2 weeks until the untransfected control cells had died. Subsequently, transfected cells were seeded (4 × 10 5 cells/mL) in 6-well plates under 500 μg/mL G418 selection pressure to produce the transfected cell pools for further analysis to determine fluorescence intensity.

| Quantitative real-time PCR analysis
Genomic DNA and total RNA were extracted according to the previous studies. 12

| Statistical analysis
All data were obtained from at least three independent experiments and were analysed using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). Data are reported as the mean ± SD. Group means were compared using single-factor analysis of variance, and t tests were used for pairwise comparisons. Differences with P < 0.05 were considered statistically significant.

| Transgene expression levels in stably transfected cells
The CHO cells were transfected by the vector containing MAR and without MAR, stably transfected clones were obtained under G418 screening pressure. The expression of eGFP in stably transfected cells was detected by flow cytometry ( Figure 1A). The results showed that the mean fluorescence intensity (MFI) of eGFP containing MAR-3 and MAR-7 was 2.79-and 2.92-fold compared with the control vector respectively ( Figure 1B), suggesting two MAR elements can increase the transgene expression level of stably transfected cells.

| qRT-PCR analysis
To investigate the correlation between eGFP activity and its mRNA levels, we performed qRT-PCR with cDNAs prepared from CHO cells

| DISCUSSION
In the present study, we investigated the effects of MAR-3 and MAR-7 on transgene expression in CHO cells using eGFP reporter gene. We found that the two MARs vectors could improve the transfection efficiency and the expression level of eGFP reporter gene and mAb compared with the control vector.
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. According to a previous study, different MAR combination influence MAR activity with respect to increasing transgene expression. 8,13 In this study, we found that the increase in eGFP gene expression level is related to the increase in the number of copies of transgenic. It was also found that the activity of eGFP was closely related to the level of mRNA containing MAR expression vector.
In conclusion, in this study, we first identified two effective MAR elements, which can improve the expression level of recombination F I G U R E 2 Analysis of the relative eGFP mRNA levels and eGFP copy number in stably transfected cell pools. Fluorescent quantitative PCR was used to measure relative eGFP mRNA levels and gene copy numbers. The 2 −ΔΔCt method was used to calculate relative values. The mRNA levels and eGFP gene copy numbers were normalized to the control whose value was set to 1. Three independent experiments were performed in this study. Standard error of the mean (SEM) is indicated (*P < 0.05) protein, and the rate of positive colonies in CHO cells. This effect may be related to the increase in transgene copy number, and the activity of recombinant protein is closely related to the level of mRNA containing MAR vector.