Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells

Wang, Fang; Wang, Tianyun; Tang, Yuanyuan; Zhang, Junhe; Yang, Xinyue

doi:10.1016/j.gene.2012.03.049

Cited by 33 publications

(21 citation statements)

References 25 publications

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“…It was previously reported that both the MAR sequence [28]–[31] and the IR sequence [32] enhance expression from cis -linked genes. Our results showed that antibody expression from the ß(−) plasmid set, which contained only MAR (CN59-4; Figure?>;6), was lower than that from the β(+) set, which contained both IR and MAR (CN59-1).…”

Section: Resultsmentioning

confidence: 99%

Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

et al. 2012

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Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

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Section: Resultsmentioning

confidence: 99%

Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

et al. 2012

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“…MARs have been studied extensively to determine their roles in gene expression, chromatin organization and DNA replication. Previous studies have suggested that MAR elements could enable more effective and more stable gene expression in CHO cells . MAR1 from CHO cells improves the level and stability of gene expression in CHO‐K1 cells, and these effects are stronger than those of the chicken lysozyme MAR.…”

Section: Discussionmentioning

confidence: 99%

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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“…Furthermore, unrelated sequence with similar in length to IR could not support the gene amplification [10]. On the other hand, there were reports that MAR [11]–[14], IR [15], anti-repressor elements [16] or chromatin opening elements [17] enhanced expression from the flanking target gene, and it was applied to the recombinant protein production. It was suggested that these sequences reduced the effect of heterochromatin that might flank the chromosomal integration site.…”

Section: Introductionmentioning

confidence: 98%

Efficient Recombinant Production in Mammalian Cells Using a Novel IR/MAR Gene Amplification Method

Araki

Hamafuji²,

Noguchi

et al. 2012

PLoS ONE

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We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone.

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Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells

Cited by 33 publications

References 25 publications

Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

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

Efficient Recombinant Production in Mammalian Cells Using a Novel IR/MAR Gene Amplification Method

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