MAR elements and transposons for improved transgene integration and expression

Ley, Déborah; Fourn, Valérie Le; Girod, Pierre‐Alain; Regamey, Alexandre; Mermod, Nicolas

doi:10.1016/j.nbt.2014.05.945

Cited by 7 publications

(10 citation statements)

References 17 publications

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“…Matrix attachment regions can increase expression levels of the transgene in stably transfected CHO cells [6][7][8][9][10][11][12]. However, the characteristics and mechanism of MARs function have not been elucidated, and further studies are needed to develop improved methods for transgene expression.…”

Section: Discussionmentioning

confidence: 99%

“…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]. However, some reports have shown that S/MARs have inconsistent effects on transgene expression [16][17][18].…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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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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“…There are two types of vectors: integrated and non-integrated vectors. Integrated vectors facilitate the insertion of mutations, and may be responsible for diseases arising as a result of the position of the inserted gene (Buceta et al, 2011;Ley et al, 2013). Ideal non-integrated vectors present no potential risk in gene therapy, are stable through mitosis, and express high levels of the transgene (Girod et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells

et al. 2015

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ABSTRACT. Preliminary studies have suggested that a characteristic element of the matrix attachment region (MAR) in human interferon-β mediates the adhesion of vectors to Chinese hamster ovary (CHO) cells. In this study, we investigated if vector adhesion increased nerve growth factor (NGF) expression in CHO cells. The MAR characteristic element sequence of human interferon-β was inserted into the multiple-cloning site of the pEGFP-C1 vector. The target NGF gene was inserted upstream of the MAR characteristic element sequence to construct the MAR/ NGF expression vector. The recombinant plasmid was transfected into CHO cells and stable monoclonal cells were selected using G418. NGF mRNA and protein expression was detected by reverse transcriptasepolymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Plasmid reduction experiments were used to determine the state of transfected plasmid in mammalian cells. 9191-9199 (2015) into the vector increased NGF expression levels in CHO cells (1.93-fold) compared to the control. The recombinant plasmid expressing the MAR sequence was digested into a linear space vector. The inserted MAR and NGF sequences were consistent with those inserted into the plasmid before recombination. Therefore, we concluded that the MAR characteristic element mediates vector adhesion to CHO cells and enhances the stability and efficiency of the target gene expression.

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“…The use of the MAR, developed in recent years, can effectively overcome exogenous gene inactivation. Many studies have focused on increasing the expression of exogenous genes using nuclear MARs (Klehr et al, 1991;Chavali et al, 2011;Harraghy et al, 2011;Jin et al, 2012;Koirala et al, 2013;Ley et al, 2013). MAR sequences may increase the production of recombinant proteins (Harraghy et al, 2012), and β-globin MAR can improve the stable genomic expression of the Sleeping Beauty transposon (Sjeklocha et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Regulating effects of insertion direction of matrix attachment regions on transgenic expression in stably transformed Chinese hamster ovary cells

Wang¹,

Zhang²,

Wang³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. We investigated the effects of different directions of insertion of matrix attachment region (MAR) sequences on transgenic expression in stably transformed Chinese hamster ovary (CHO) cells. The MAR sequences were inserted in forward or reverse directions into the expression vectors, and transfected into CHO cells. The expression of the chloramphenicol acetyltransferase (CAT) reporter gene and the relative copy numbers of the CAT gene were analyzed. The CAT gene expression levels in the vector with the MAR sequence inserted in the forward or reverse directions increased compared with expression without the MAR sequence. The relative copy numbers of the CAT gene with MAR sequenced vectors inserted in the reverse and forward directions were lower, than in the control group. The direction of insertion of MAR sequences had no significant effect on expression levels. The expression levels were not proportional to the copy numbers

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MAR elements and transposons for improved transgene integration and expression

Abstract: Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable.

Cited by 7 publications

References 17 publications

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

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

Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells

Regulating effects of insertion direction of matrix attachment regions on transgenic expression in stably transformed Chinese hamster ovary cells

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