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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos
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Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

机译：锌指核酸酶介导的报告基因的定向插入，用于海胆胚胎中基因表达的定量成像

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摘要

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme Fokl and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEtsi gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEtsi with a targeting donor construct that contained ~1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form, of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone non-homologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEtsi expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.
机译：要了解复杂的生物系统，例如多细胞生物的发育，表征基因表达动态非常重要。但是，目前还没有在多细胞模型系统中靶向性插入报告基因和定量成像的通用技术。最近，在几种模型中已经报道了使用锌指核酸酶（ZFN）进行基因组编辑。 ZFN由具有限制酶Fok1的核酸酶结构域的锌指DNA结合阵列组成，并有助于靶向转基因插入。在这项研究中，我们成功地将GFP报告基因盒插入到海胆Hemicentrotus pulcherrimus的HpEtsi基因位点中。我们通过向鸡蛋中注射一对HpEtsi ZFN来实现这一插入，该ZFN具有靶向供体构建体，该构建体包含〜1-kb同源臂和2A-组蛋白H2B-GFP盒。我们通过靶向供体构建体的原位线性化和共显性显性阴性形式的HpLig4 mRNA的mRNA引入，提高了ZFN介导的靶向转基因插入的效率，HpLig4编码错误所需的DNA连接酶IV的H. pulcherrimus同源物。 -倾向于非同源末端连接。我们在发育过程中在活胚胎中的单细胞水平上测量了GFP的荧光强度，发现原代间充质细胞之间HpEtsi表达存在差异。这些发现证明了ZFN介导的靶向转基因插入在活海胆胚胎发育期间能够量化内源基因表达水平的可行性。

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    Hiroshi Ochiai; Naoaki Sakamoto; Kazumasa Fujita; Masatoshi Nishikawa; Ken-ichi Suzuki; Shinya Matsuura; Tatsuo Miyamoto; Tetsushi Sakuma; Tatsuo Shibata; Takashi Yamamoto;

  • 作者单位

    Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima 734-8553,Japan;

    Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan;

    Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan;

    Laboratory for Physical Biology, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Hyogo 650-0047, Japan;

    Center for Marine Environmental Studies, Ehime University, Matsuyama 790-8577, Japan;

    Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima 734-8553,Japan;

    Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima 734-8553,Japan;

    Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan;

    Laboratory for Physical Biology, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Hyogo 650-0047, Japan;

    Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    live imaging; quantitative biology;

    机译：实时成像;定量生物学;

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