Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

Chun-hua, Zhao; Guo, Xiao; Chen, Si-Jia; Li, Changzheng; Yang, Yun Kang; Zhang, Junhe; Chen, Shao-Nan; Jia, Yanjie; Wang, Tianyun

doi:10.1038/srep42805

Cited by 38 publications

(33 citation statements)

References 35 publications

(50 reference statements)

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“…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.…”

Section: Discussionsupporting

confidence: 92%

“…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.…”

Section: Discussionmentioning

confidence: 92%

“…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 …”

Section: Discussionmentioning

confidence: 94%

“…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 …”

Section: Introductionmentioning

confidence: 99%

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Two human MARs effectively increase transgene expression in transfected CHO cells

Chun-hua

Lin

et al. 2018

J Cellular Molecular Medi

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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.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Two human MARs effectively increase transgene expression in transfected CHO cells

Chun-hua

Lin

et al. 2018

J Cellular Molecular Medi

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“…In previous studies, the eGFP gene was initially used to study the effect of MAR on transgene expression in transfected CHO cells, and genes of interest (GOI), such as antibodies and other proteins, were further studied. GOI expression levels were consistent with the levels of eGFP . We will investigate some the production of antibodies and other biopharmaceutical proteins in future studies.…”

Section: Discussionsupporting

confidence: 60%

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

Tian

Wang

et al. 2017

J Cellular Molecular Medi

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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.

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Engineered and Natural Promoters and Chromatin‐Modifying Elements for Recombinant Protein Expression in CHO Cells

Romanova

Noll

2017

Biotechnology Journal

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Promoters play a pivotal role in integrating and processing the signals related to transcription initiation. Strong natural viral promoters, such as hCMV or SV40E, have been routinely employed to achieve a high rate of gene expression in ubiquitously used Chinese hamster ovary (CHO) cells. However, viral promoters are susceptible to epigenetic silencing and lack precise regulation levers. This has paved the way to more sensible control elements: endogenous, inducible, and synthetic promoters. In this review we summarize and discuss the use of natural viral, mammalian, and endogenous promoters, as well as recent advances in synthetic promoters and inducible systems for protein expression in CHO cells. Not only the level of transcription, but its long-term stability is crucial for recombinant protein production. Epigenetic chromatin-modifying elements, such as ubiquitously acting chromatin opening elements (UCOEs), matrix attachment regions (MARs), insulators and stabilizing anti-repressors (STARs) significantly improve transcription levels over extended cultivation time and are also discussed here. This review provides up-to date information to facilitate the choice of a suitable promoter and adjacent chromatin-modifying elements to maximize transgene expression as well as ensure long-term expression stability in CHO cell culture.

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Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

Cited by 38 publications

References 35 publications

Two human MARs effectively increase transgene expression in transfected CHO cells

Two human MARs effectively increase transgene expression in transfected CHO cells

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

Engineered and Natural Promoters and Chromatin‐Modifying Elements for Recombinant Protein Expression in CHO Cells

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