Scaffold/matrix attachment regions from <scp>CHO</scp> cell chromosome enhanced the stable transfection efficiency and the expression of transgene in <scp>CHO</scp> cells

Chang, Ming Shi; Liu, Ruijie; Jin, Qingzhe; Liu, Yuanfa; Wang, Xingguo

doi:10.1002/bab.1204

Cited by 25 publications

(11 citation statements)

References 23 publications

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“…Human CSP‐B MAR (hu‐MAR) (GenBank No. M62716), MAR‐6 from CHO cells (MAR1) (GenBank No. NW_003613799.1, position 724006–725949) and MAR from the DHFR intron (MAR2) (GenBank No.…”

Section: Methodsmentioning

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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“…Human CSP‐B MAR (hu‐MAR) (GenBank No. M62716), MAR‐6 from CHO cells (MAR1) (GenBank No. NW_003613799.1, position 724006–725949) and MAR from the DHFR intron (MAR2) (GenBank No.…”

Section: Methodsmentioning

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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“…Scaffold/matrix attachment regions (S/MARs) can block transgene silencing [6][7][8] and increase transgene expression levels and stability in host cells [9][10][11][12][13][14][15]. In addition, S/MARs can also reduce variations in transgene expression among different cells to some extent, and the rate of transgene genomic integration can be increased [11].…”

mentioning

confidence: 99%

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

et al. 2017

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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.

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“…Moreover, MARs can shield transgenes from position, superposition, and distance effects to enhance their expression. 17,18,[22][23][24][25][26] The effects of MARs on transgene expression have been reported; however, the enhancing effects of MARs still need to be improved. In addition, commonly used natural MARs, such as MAR 1-68, X-29, and chicken lysozyme MAR, are larger DNA fragments that are inconvenient to clone and show lower transfection efficiency.…”

Section: Abstract: Cho Cell; Matrix Attachment Regions; Transgene Exmentioning

confidence: 99%

A short synthetic chimeric sequence harboring matrix attachment region/PSAR2 increases transgene expression in Chinese hamster ovary cells

Qin

Wen

Jia

et al. 2017

Bioscience, Biotechnology, and Biochemistry

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A chimeric DNA fragment containing an interferon-beta matrix attachment region (MAR) and an immunoglobulin MAR (PSAR2) was synthesized. PSAR2 was cloned into the upstream or downstream region of an enhanced green fluorescent protein (eGFP) expression cassette in a eukaryotic vector, which was then transfected into CHO cells. The results showed that PSAR2 did not effectively increase transgene expression when it was cloned into the upstream region of the eGFP expression cassette. However, when inserted downstream of the eGFP expression cassette, PSAR2-enhanced transient transgene expression and significantly increased the numbers of stably transfected cells compared with the control vector. Additionally, PSAR2 significantly increased eGFP copy numbers as compared with the control vector. PSAR2 could significantly enhance transgene expression in CHO cells according to the position in the vector and increased transgene copy numbers. We found a short chimeric sequence harboring two MARs effectively increased transgene expression in CHO cells.

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Scaffold/matrix attachment regions from CHO cell chromosome enhanced the stable transfection efficiency and the expression of transgene in CHO cells

Cited by 25 publications

References 23 publications

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

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

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

A short synthetic chimeric sequence harboring matrix attachment region/PSAR2 increases transgene expression in Chinese hamster ovary cells

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