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

Authors

  • Satoshi Watanabe,

    Corresponding author
    1. Aminal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukuba, Ibaraki, 305-8602, Japan
      *Correspondence: E-mail: kettle@affrc.go.jp
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  • Sachiko Watanabe,

    1. Aminal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukuba, Ibaraki, 305-8602, Japan
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  • Naoaki Sakamoto,

    1. Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8526, Japan
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  • Masahiro Sato,

    1. The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa, 259-1193, Japan
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      Frontier Science Research Center, Kagoshima University, Korimoto 1-21-40, Kagoshima 890–0065, Japan.
  • Koji Akasaka

    1. Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8526, Japan
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      bMisaki Marine Biological Station, Graduate School of Science, University of Tokyo, 1024, Koajiro, Misaki, Miura, Kanagawa 238–0225, Japan.

  • Communicated by: Shinichi Aizawa

  • a

    Present addresses: Frontier Science Research Center, Kagoshima University, Korimoto 1-21-40, Kagoshima 890–0065, Japan. bMisaki Marine Biological Station, Graduate School of Science, University of Tokyo, 1024, Koajiro, Misaki, Miura, Kanagawa 238–0225, Japan.

*Correspondence: E-mail: kettle@affrc.go.jp

Abstract

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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