An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells

Nagaya, Shingo; Yoshida, Kazuya; Kato, Ko; Akasaka, Koji; Shinmyo, Atsuhiko

doi:10.1007/s004380100448

Cited by 31 publications

(5 citation statements)

References 27 publications

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“…The Ars ‐insulator was also shown to be useful for avoiding transgene silencing in transgenics (Takada et al . 2000; Nagaya et al . 2001; Hino et al .…”

Section: Discussionmentioning

confidence: 99%

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Functional analysis of the sea urchin‐derived arylsulfatase (Ars)‐element in mammalian cells

Watanabe

Sakamoto

et al. 2006

Genes to Cells

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An insulator is a DNA sequence that has both enhancer-blocking activity, through its ability to modify the influence of neighboring cis -acting elements, and a barrier function that protects a transgene from being silenced by surrounding chromatin. Previously, we isolated and characterized a 582-bp-long element from the sea urchin arylsulfatase gene ( Ars ). This Ars-element was effective in sea urchin and Drosophila embryos and in plant cells. To investigate Ars -element activity in mammalian cells, we placed the element between the cytomegalovirus enhancer and a luciferase (luc) expression cassette. In contrast to controls lacking the Ars -element, NIH3T3 and 293T cells transfected with the element-containing construct displayed reduced luciferase activities. The Ars -element therefore acts as an enhancer-blocking element in mammalian cells. We assessed the barrier activity of the Ars -element using vectors in which a luc expression cassette was placed between two elements. Transfection experiments demonstrated that luc activity in these vectors was approximately ten-fold higher than in vectors lacking elements. Luc activities were well maintained even after 12 weeks in culture. Our observations demonstrate that the Ars -element has also a barrier activity. These results indicated that the Ars -element act as an insulator in mammalian cells.

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“…The Ars ‐insulator was also shown to be useful for avoiding transgene silencing in transgenics (Takada et al . 2000; Nagaya et al . 2001; Hino et al .…”

Section: Discussionmentioning

confidence: 99%

“…We recently found that a 582‐bp fragment, located upstream of the Ars gene, functions as an insulator in both sea urchin and Drosophila embryos and in plants (Takada et al . 2000; Nagaya et al . 2001).…”

Section: Introductionmentioning

confidence: 99%

Functional analysis of the sea urchin‐derived arylsulfatase (Ars)‐element in mammalian cells

Watanabe

Sakamoto

et al. 2006

Genes to Cells

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“…Non-plant insulator sequences that, at least to some extent, act as insulators in transgenic Arabidopsis include UASrpg isolated from Ashbya gossypii , BEAD1c from H. sapiens [ 34 ], and Fab-7 and gypsy from Drosophila [ 35 , 36 ]. In transgenic tobacco, an insulator from the sea urchin Hemicentrotus pulcherrimus exhibited enhancer-blocking activity [ 37 ]. The notion that insulator function is conserved across kingdoms is further supported by the finding that binding sites for insulator proteins Rap2, CTCF and Su(Hw) were functional in transgenic Arabidopsis as a part of the insulator heterologous sequences from A. gossypii , H. sapiens and D. melanogaster.…”

Section: The Existence Of Plant Insulatorsmentioning

confidence: 99%

Insulators in Plants: Progress and Open Questions

Kurbidaeva

Purugganan

2021

Genes

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The genomes of higher eukaryotes are partitioned into topologically associated domains or TADs, and insulators (also known as boundary elements) are the key elements responsible for their formation and maintenance. Insulators were first identified and extensively studied in Drosophila as well as mammalian genomes, and have also been described in yeast and plants. In addition, many insulator proteins are known in Drosophila, and some have been investigated in mammals. However, much less is known about this important class of non-coding DNA elements in plant genomes. In this review, we take a detailed look at known plant insulators across different species and provide an overview of potential determinants of plant insulator functions, including cis-elements and boundary proteins. We also discuss methods previously used in attempts to identify plant insulators, provide a perspective on their importance for research and biotechnology, and discuss areas of potential future research.

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“…However, despite recent advances in gene-editing technologies (Nishizawa-Yokoi et al 2012;Osakabe et al 2010), the recombination rate is relatively low to produce transgenic plants. Another approach to reduce the variation of transgene expressions is developed by utilizing an insulator, which is chromatin boundary elements, such as matrix-attachment regions (MARS), to prevent mislocalization of transgenes in heterochromatin (Breyne et al 1992;Nagaya et al 2001). Though it was reported that the vector with an insulator at the borders of T-DNA indeed reduced the variability in β-glucuronidase (GUS) activities (Nagaya et al 2001), the copy number of a transgene is still uncontrollable.…”

Section: Introductionmentioning

confidence: 99%

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences

Takita

Yoshida

Hanano

et al. 2021

Plant Biotechnology

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Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.

show abstract

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells

Cited by 31 publications

References 27 publications

Functional analysis of the sea urchin‐derived arylsulfatase (Ars)‐element in mammalian cells

Functional analysis of the sea urchin‐derived arylsulfatase (Ars)‐element in mammalian cells

Insulators in Plants: Progress and Open Questions

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences

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