Improved recombinant gene expression in CHO cells using matrix attachment regions

Kim, Jong-Mook; Kim, Jung-Seob; Park, Doo-Hong; Kang, Ho Sung; Yoon, Jaeseung; Baek, Kwanghee; Yoon, Yeup

doi:10.1016/j.jbiotec.2003.09.015

Cited by 120 publications

(74 citation statements)

References 30 publications

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“…Finally, the MAR elements might favor integration at permissive loci within the chromosome, or they may increase the number of transgene copies integrated. With regard to the latter possibility, Kim et al (2004) have observed higher transgene copy numbers when these are co-transfected for the b-globin MAR. In the present study, higher transgene copy numbers were also observed on average in the clones obtained by transfecting the transgene with the MAR in cis and in trans when compared to clones transfected with MAR in cis or trans only, which in turn had more transgene copies than the clones generated without the MAR element.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, the use of these boundary or insulator elements for the production of recombinant proteins has raised considerable interest. Several studies demonstrated the advantage of using them to increase recombinant protein synthesis in mammalian cell lines of biotechnological interest (Kim et al, 2004;Zahn-Zabal et al, 2001), or for transgenesis and gene therapies in vivo (Agarwal et al, 1998;Allen et al, 1996;Castilla et al, 1998). For instance, scaffold or matrix attachment regions (SARs or MARs, collectively referred to as S/MAR elements) were shown to improve the probability of isolating a clone exhibiting the desired level of expression for the production of a recombinant protein and/ or to increase the stability of production.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Girod

Zahn-Zabal

Mermod

2005

Biotechnol. Bioeng.

104

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Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/ MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Girod

Zahn-Zabal

Mermod

2005

Biotechnol. Bioeng.

104

View full text Add to dashboard Cite

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“…However, some studies have suggested that the effect of MARs on transgenic expression depends on transgenic copy numbers (Kim et al, 2004;Oh et al, 2005). Our results indicate that the expression level of the MAR CAT gene inserted in the reverse direction is lower than that of the MAR inserted in the forward direction.…”

Section: Discussionmentioning

confidence: 47%

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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“…Therefore, optimizing the foreign genes at the construction level has become the best approach to ensure high degree of protein production. Approaches such as DNA elements integration [5], promoter element integration [6] and scaffold/matrix attachment region integration [7] into expression vector system have proved to be effective. Another factor that has significant effect towards recombinant protein production and has been applied extensively is insertion of signal sequence coding region together with transgene sequence which facilitates in mRNA export to reticulum endothelium for processing [8].…”

Section: Introductionmentioning

confidence: 99%

“…The most favorable locus for introducing MAR is proposed to be upstream of promoter and enhancer region in expression vector [9]. Even the proper mechanisms by which MAR elements contribute to increase protein being expressed still remain unclear, several studies have reported on the enhancement of transgene expression with copy number-dependent or positionindependent properties of MAR elements [7,14,15]. Recent finding has reported that combination of MAR element with mammalian replication initiation region in CHO DG44 cells significantly initiated gene amplification effectively and spontaneously increased gene copy number [16].…”

Section: Introductionmentioning

confidence: 99%

Integrated Analytical Approaches Towards Fungal Natural Products Discovery, Pathway Elucidation and Synthetic Biology

Larsen

2013

TOPROCJ

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Abstract:The emergence of monoclonal antibody specific to human growth factor receptor marked the greatest achievement in human fight against cancer. By utilizing a specific antibody, cancer treatment nowadays is getting more specific and efficient. Despite its achievement, the main drawback of antibody therapy remains at its production level. Low yield and transient expression of antibody by mammalian host cells are among the problems discovered during antibody production. Many strategies have been implied to increase yield and stability of protein including improvement of vector expression system with DNA elements integration as well as manipulating the bioreactor environment to increase cell density and attain more products. Our study is currently focused on constructing a high expression vector system using matrix attachment region (MAR) element in targeted therapeutic monoclonal antibody production. In this study, a nuclear halo formation has been successfully optimized for CHO cells. A minimum time of 8 minutes is required for maximum nuclear halo formation, thus exposing the most significant sites containing MAR element in CHO genome. This preliminary step is crucial due to the fact that the nuclear halo formation and MAR elements isolation are specific for different types of cells. Subsequent works are currently being carried out to isolate the MAR element from CHO cells based on the time determined from nuclear halo formation. This work is going to be distinctive from previous studies as the MAR element will be predicted from the host cells genome. The mechanism of which the integrated functional MAR element can increase the antibody production even at random position is through up-regulation of gene transcription by adopting a DNA loop structure of nucleosomes that open the structure for specific transcription factor binding.

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Improved recombinant gene expression in CHO cells using matrix attachment regions

Cited by 120 publications

References 30 publications

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines

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

Integrated Analytical Approaches Towards Fungal Natural Products Discovery, Pathway Elucidation and Synthetic Biology

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