The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.

Phi‐van, Loc; Kries, Jens Peter von; Ostertag, Wolfram; Strätling, Wolf H.

doi:10.1128/mcb.10.5.2302

Cited by 259 publications

(95 citation statements)

References 37 publications

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“…A transgene flanked by S/MAR elements may therefore constitute an autonomous chromatin domain whose expression would remain independent of the adjacent chromosomal environment. Consistent with this view, S/ MARs have been shown to increase the expression of adjacent transgenes when co-inserted into a chromosomal environment (Kalos and Fournier, 1995;Klehr et al, 1991;Phi-Van et al, 1990;Poljak et al, 1994). …”

Section: Introductionmentioning

confidence: 68%

“…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%

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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.

103

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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: Introductionmentioning

confidence: 68%

Section: Stable Gfp Expression In Cho Cells With the Marmentioning

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.

103

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“…MARs are AT-rich DNA sequences that bind to the nuclear matrix in vitro and are thought to organize chromosomal DNA into loop domains (Bonifer et al, 1990;Bode et al, 1996;Martelli et al, 2002). Early experiments in animal cells indicated that MARs increased transgene expression and decreased transformantto-transformant variability in transgene expression (Stief et al, 1989;Bonifer et al, 1990;Phi-Van et al, 1990). Subsequent work in plant systems also demonstrated an increase in transgene expression and/or a reduction in variability (Breyne et al, 1992;Allen et al, 1993Allen et al, , 1996Mlynarova et al, 1994Mlynarova et al, , 1995Han et al, 1997;Liu and Tabe, 1998;Ü lker et al, 1999;Vain et al, 1999;Cheng et al, 2001;Mendu et al, 2001;Fukuda and Nishikawa, 2003;Mankin et al, 2003;Butaye et al, 2004;van der Geest et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

2005

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Many studies in both plant and animal systems have shown that matrix attachment regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. Because histochemical assays often indicate variegated transgene expression, a question arises: Do MARs increase transgene expression by increasing the percentage of cells expressing the transgene (likelihood), by increasing the level of expression in expressing cells (magnitude), or both? To address this question, we used flow cytometry to measure green fluorescent protein (GFP) expression in individual tobacco (Nicotiana tabacum) cells from lines transformed by Agrobacterium tumefaciens. We conclude that MAR-mediated overall increases in transgene expression involve both likelihood and magnitude. On average, cell lines transformed with the Rb7 MAR-containing vector expressed GFP at levels 2.0- to 3.7-fold higher than controls. MAR lines had fewer nonexpressing cells than control lines (10% versus 45%), and the magnitude of GFP expression in expressing cells was greater in MAR lines by 1.9- to 2.9-fold. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from populations of independently transformed cell lines. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression

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“…These putative structural elements of chromatin have attracted considerable attention because of evidence for their involvement in the regulation of transcription, including stabilization and increased levels of transgene expression (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) [reviewed by Bode et al (4)]. The comapping of MARs and origins of replication and the functioning of MARs as ARS elements suggest a role for MARs in DNA replication (28)(29)(30)(31)(32)(33)(34).…”

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confidence: 99%

Characterization of Randomly-Obtained Matrix Attachment Regions (MARs) from Higher Plants^,

et al. 1999

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Matrix attachment regions (MARs) can be operationally defined as DNA fragments that bind to the nuclear matrix. We have created a library of randomly obtained MARs from tobacco (Nicotiana tobacum) by cloning DNA fragments that co-isolate with nuclear matrixes prepared by a method involving lithium diiodosalicylate. The interactions of several of the cloned MARs with nuclear matrixes were tested by an in vitro binding assay in which genomic DNA was used as competitor. Based on this assay, the MARs were classified as strong, medium, and weak binders. Examples of each of the binding classes were further studied by in vitro binding using self-and cross-competition. Estimates of dissociation constants for several MARs ranged from 6 to 11 nM and correlated inversely with binding strength. The number of binding sites per matrix for several MARs ranged from 4 × 10 5 to 9 × 10 5 and correlated directly with binding strength. We conclude that binding strength, as we have measured it, is a function of both numbers of binding sites and affinity for the sites. The tobacco MARs were sequenced and analyzed for overall AT content, for distribution of AT-rich regions, and for the abundance of several MAR-related motifs. Previously identified MAR motifs correlate to various degrees with binding strength. Notably, the Drosophila topoisomerase II motif does not correlate with binding strength of the tobacco MARs. A newly identified motif, the "90%AT Box," correlates better with binding strength than any of the previously identified motifs we investigated.The idea that chromatin is organized into loop domains is as old as the original observation of loops in lampbrush chromosomes by Flemming in 1882 (1, 2). A reincarnation of that idea came from the electron micrographs of Paulson and Laemmli (3), which showed loops of DNA attached at their bases to the proteinaceous scaffold of metaphase chromosomes. A natural extension of the observations of Paulson and Laemmli is that the DNA-chromosome scaffold interaction is not random. Indeed, abundant evidence has been presented for interactions between specific DNA fragments and the metaphase chromosome scaffold and the interphase nuclear matrix [reviewed in Bode et al. (4,5)]. The specific DNA sequences have been termed MARs (matrix associated regions) (6) or SARs (scaffold attachment regions) (7,8). Both terms apparently describe the same biological entity (9). We use the term "MAR" because of its prevalence in the literature (10).MARs have a typical size of around 1 kb and have been postulated to form the anchorage points of torsionally constrained loop domains ranging in size from a few kb to more than 100 kb [reviewed by Gasser et al. (11) and Bode et al. (4,5)]. These putative structural elements of chromatin have attracted considerable attention because of evidence for their involvement in the regulation of transcription, including stabilization and increased levels of transgene expression (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) [reviewed by Bode e...

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The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.

Cited by 259 publications

References 37 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

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

Characterization of Randomly-Obtained Matrix Attachment Regions (MARs) from Higher Plants^,

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The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.

Cited by 259 publications

References 37 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

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

Characterization of Randomly-Obtained Matrix Attachment Regions (MARs) from Higher Plants,

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Characterization of Randomly-Obtained Matrix Attachment Regions (MARs) from Higher Plants^,