A chimeric HS4 insulator–scaffold attachment region enhances transgene expression in transfected Chinese hamster ovary cells

Abstract

Chinese hamster ovary ( CHO ) cells are one of the most commonly used expression systems for the production of recombinant proteins but low levels of transgene expression and transgene silencing are frequently encountered. Epigenetic regulatory elements such as the chicken β‐globin locus control region hypersensitive site 4 (HS4) and scaffold/matrix attachment regions (S/MARs) have positive effects on transgene expression. In this study, a chimeric HS 4‐ SAR was cloned upstream or downstream of an enhanced green fluorescent protein ( eGFP ) expression cassette in a eukaryotic vector, and the resulting vectors were transfected into CHO cells. eGFP was detected by flow cytometry. Real‐time quantitative PCR (qPCR) was used to determine copy numbers of the stably transfected cells. And fluorescence in situ hybridization ( FISH ) was used to detect the status of vector in the host cell chromosome. The results showed that HS 4‐ SAR positioned downstream of the expression cassette could enhance eGFP expression by 4.83‐fold compared with the control vector. There may not be a relationship between transgene copy number and gene expression level. HS 4‐ SAR did not appear to alter the integration of the transgene into the host cell chromosome or its position in the chromosome. We found a synthetic chimeric HS 4‐ SAR positively increased transgene expression in CHO cells.

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 …”

Identification of consensus sequence from matrix attachment regions and functional analysis of its activity in stably transfected Chinese hamster ovary cells

“…However, low expression levels of recombinant proteins or transgene silencing caused by positional effects remain one of the most significant challenges faced in recombinant protein production. 8,9 To achieve high and stable production of therapeutic proteins, many strategies have been adopted in recombinant mammalian cells, such as the optimization of expression vectors, development of serum-free media medium, and genome editing using gene engineering. [10][11][12][13] Among these, expression vector optimization seems to be the most effective means of achieving higher and more stable transgene expression in transfected CHO cells.…”