Development of stable cell lines for production or regulated expression using matrix attachment regions

Zahn-Zabal, Monique; Kobr, Michel; Girod, Pierre‐Alain; Imhof, Markus O.; Chatellard, Philippe; Jesús, María de; Wurm, Florian M.; Mermod, Nicolas

doi:10.1016/s0168-1656(00)00423-5

Cited by 136 publications

(89 citation statements)

References 34 publications

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“…The MAR element of pPAG1 was inserted in pSV40EGFPcontrol to generate pPAG01SV40EGFP, thus containing successively the MAR element, the SV40 promoter, the eGFP coding sequence, and the SV40 enhancer. The immunoglobulin heavy and light chain expression vectors driven by the SV40 early promoter/ enhancer alone (pMZ37, pMZ59) or by the human CMV promoter (pMZ36, pMZ57) are as described elsewhere (Miescher et al, 2000;Zahn-Zabal et al, 2001). Derivatives containing one MAR inserted upstream of the SV40 enhancer/promoter were created by cloning the BamH1-Xba I MAR fragment in plasmids pMZ59 and pMZ37 linearized with EcoRI and BamHI respectively, after first blunting the XbaI site of the MAR fragment and EcoRI site of pMZ59 and pMZ37 with Pfu.…”

Section: Plasmidsmentioning

confidence: 99%

“…Pools of stable CHO cells expressing GFP or anti-Rhesus D immunoglobulin were obtained by transfection with polyethyleneimine (PEI) as described in Boussif et al (1995) and Zahn-Zabal et al (2001), or with LipofectAMINE 2000 (Invitrogen, Inc., Basel, Switzerland), as recommended by the manufacturer. Cells were seeded in 6-well plates at 500-750,000 cells/well and allowed to attach overnight.…”

Section: Cho Cell Culture and Transfectionmentioning

confidence: 99%

“…The chicken lysozyme MAR element has been reported to mediate elevated transgene expression when introduced in cis into the expression vector (Phi- Van et al, 1990;Stief et al, 1989), or when co-transfected in trans on a distinct plasmid (Zahn-Zabal et al, 2001). In this study, we systematically analyzed the use of the MAR element in such experimental settings, with the perspective of producing recombinant proteins using CHO cell lines.…”

Section: Stable Gfp Expression In Cho Cells With the Marmentioning

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%

“…The chicken lysozyme 5 0 MAR was identified as one of the most active sequence in a previous exploratory study that compared the effect of various chromatin structure regulatory elements on transgene expression (Zahn-Zabal et al, 2001). This element was shown to increase the levels of regulated or constitutive transgene expression in various mammalian cell lines.…”

Section: Introductionmentioning

confidence: 99%

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

Girod

Zahn-Zabal

Mermod

2005

Biotechnol. Bioeng.

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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: Plasmidsmentioning

confidence: 99%

Section: Cho Cell Culture and Transfectionmentioning

confidence: 99%

Section: Stable Gfp Expression In Cho Cells With the Marmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Girod

Zahn-Zabal

Mermod

2005

Biotechnol. Bioeng.

Self Cite

104

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Chinese Hamster Ovary Cells, Recombinant Protein Production

Hacker

Chenuet

Wurm

2010

Encyclopedia of Industrial Biotechnology

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Recombinant therapeutic proteins represent a major class of biopharmaceuticals. They are produced in various hosts including bacteria, yeast, cultivated mammalian and plant cells, and transgenic plants and animals. Chinese hamster ovary (CHO) cells are the most widely used mammalian host for their production. Currently, about one‐third of all approved recombinant therapeutic proteins are produced in this cell line. This article is a summary of the molecular and cellular processes involved in therapeutic protein production with an emphasis on the generation and cultivation of recombinant CHO cell lines.

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Transcription, Translation, and the Control of Gene Expression in Mammalian Cells

Mullick¹,

Massie²

2010

Encyclopedia of Industrial Biotechnology

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The last few years have seen rapid progress in the field of gene expression, and at least part of the motivation has come from the domain of biotechnology, wherein, recombinant proteins have become important players in therapeutics. Depending upon the stage of development, there may be a need for small quantities of several related proteins or extremely large quantities of a restricted number of proteins. In either case, protein expression systems are essential. It has been possible to develop efficient expression systems because of the phenomenal increase in our understanding of the molecular mechanism that are involved in gene expression. Of interest is the fact that recent findings are not limited to defining further details of known processes, certain advances, such as the role of chromatin in DNA biochemistry and RNA silencing, have revolutionized the way we think about gene expression.

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Development of stable cell lines for production or regulated expression using matrix attachment regions

Cited by 136 publications

References 34 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

Chinese Hamster Ovary Cells, Recombinant Protein Production

Transcription, Translation, and the Control of Gene Expression in Mammalian Cells

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