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2005 Fiscal Year Final Research Report Summary

Development of an effective method for production of cattle that contain enhanced healthy polyunsaturated fatty acids.

Research Project

Project/Area Number 15380196
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied animal science
Research InstitutionKinki University

Principal Investigator

SAEKI Kazuhiro  Kinki University, School of Biology-Oriented Science and Technology, Professor, 生物理工学部, 教授 (10298937)

Co-Investigator(Kenkyū-buntansha) HOSOI Yoshihiko  Kinki University, School of Biology-Oriented Science and Technology, Professor, 生物理工学部, 教授 (70192739)
MATSUMOTO Kazuya  Kinki University, School of Biology-Oriented Science and Technology, Professor, 生物理工学部, 教授 (20298938)
MITANI Tasuku  Kinki University, School of Biology-Oriented Science and Technology, Associate professor, 先端技術総合研究所, 助教授 (10322265)
Project Period (FY) 2003 – 2005
Keywordsα-linolenic acid / ω3 fatty acid desaturase / bovine fibroblast cells / mouse 3T3-L1 cells / codon usage / luciferase expression vector / cloned embryos / DNA methylation
Research Abstract

The purpose of this study is to produce cattle containing healthy polyunsaturated fatty acids that are not biosynthesized by mammals. To achieve this purpose, we developed a method to produce transgenic cattle carrying a gene for a fatty acid desaturase taken from plants that are able to newly biosynthesize the polyunsaturated fatty acids.
We obtained an FAD3 cDNA which encoded an ω3 fatty acid desaturase from flax seeds that contained the highest rate of α-linolenic acid (18:3n-3) contents in land plants. New synthesis of 18:3n-3 was observed when the cDNA was introduced into yeast and examined their fatty acid composition. Furthermore, a transfection method of a foreign gene stably into mammalian primary cultured cell was established. Using this method, pβ-act/FAD3/IRES/EGFP(neor) was transfected into bovine fibroblast cells and mouse 3T3-L1 cells, and then the functional expression was examined by determination of composition rates of 18:3n-3. The FAD3 gene derived from flax seeds ex … More pressed functionally in mammalian cultured cells, because the ratio of the 18:3n-3 increased in the transfected cells with the FAD3 gene compared with that in wild-type cells. However, the ratio in the transfected cells were low and only 0.4% (wild type : 0.2%). Therefore, the DNA sequence of the FAD3 that was a plant type was changed to the mammalian-type DNA sequence according to the mammalian codon usage. Then we obtained a new FAD3 gene optimized in mammalian cells (FAD3opt), and expression vector (pCAG/FAD3opt/IRES/EGFP(neor)) was produced. Strong expression of EGFP was observed in the mouse 3T3-L1 cells transfected with pCAG/FAD3opt/IRES/EGFP(neor). In addition, we have obtained a bovine primary fibroblast cell lines carrying pCAG/FAD3opt/IRES/EGFP(neor). We now examine the fatty acid composition in the transfected cells.
We also examined the relation of gene expression in bovine cloned embryos to their further development, to establish an effective method for production of cloned calves with the transgenic cells as donor cells. We examined gene expression in embryos reconstructed with bovine fibroblasts carrying a luciferase expression vector (pβ-act/luc+/IRES/EGFP(neor)). As a result, when intensity of LUC+ expression was examined in transgenic cloned embryos 60 hours post fusion (hpf), strongly expressed-embryos developed at a high rate. Moreover, when The embryos were then classified as being Luc-positive, mosaic, or Luc-negative depending on whether all, some or no blastomeres were luminescent, respectively, the developmental rates to the blastocyst stage of positive embryos were higher than those of mosaic embryos. Any negative embryos did not developed to blastocysts. Then, we examined DNA methylation levels in the embryos by immunostaining with a 5-methyl cytosine antibody. The methylation level of the Luc-positive embryos was lower than that of the Luc-negative embryos. These results indicated that the developmental capacity of NT embryos could be correlated positively to the gene expression ability and negatively to the DNA methylation level at 4- to 8-cell stage. Less

  • Research Products

    (28 results)

All 2006 2005 2004 2003

All Journal Article (26 results) Book (2 results)

  • [Journal Article] Early morphological nuclear events and developmental capacity of embryos reconstructed with fetal fibroblasts at the M or GI phase after intracytoplasmic nuclear injection in cattle.2005

    • Author(s)
      Ideta A
    • Journal Title

      Journal of Reproduction and Development 51

      Pages: 187-194

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Fine surface structure of bovine acrosome-intact and reacted spermatozoa observed by atomic force microscopy.2005

    • Author(s)
      Saeki K
    • Journal Title

      Journal of Reproduction and Development 51

      Pages: 293-298

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] 単為発生胚からES細胞樹立と多分化能の検討.2005

    • Author(s)
      寺村岳士
    • Journal Title

      近畿大学先端技術総合研究所紀要 10

      Pages: 69-76

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] 牛体細胞核移植胚の培養液の検討.2005

    • Author(s)
      佐伯和弘
    • Journal Title

      近畿大学先端技術総合研究所紀要 10

      Pages: 63-68

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] ジメチルスルフォオキシド添加がマウス体細胞核移植胚発生に及ぼす影響.2005

    • Author(s)
      國枝孝典
    • Journal Title

      近畿大学先端技術総合研究所紀要 10

      Pages: 53-56

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Early morphological nuclear events and developmental capacity of embryos reconstructed with fetal fibroblasts at the M or G1 phase after intracytoplasmic nuclear injection in cattle.2005

    • Author(s)
      Ideta A.
    • Journal Title

      Journal of Reproduction and Development 51

      Pages: 187-194

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Fine surface structure of bovine acrosome-intact and reacted spermatozoa observed by atomic force microscopy.2005

    • Author(s)
      Saeki K.
    • Journal Title

      Journal of Reproduction and Development 51

      Pages: 293-298

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Establishment of embryonic stem cell lines derived from mouse parthenote and confirmation of their pluripotency.2005

    • Author(s)
      Teramura T.
    • Journal Title

      Memoirs of Institute of Advanced Technology, Kinki University 10

      Pages: 69-76

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Optimization of culture medium for bovine embryos reconstructed with bovine fibroblasts.2005

    • Author(s)
      Saeki K.
    • Journal Title

      Memoirs of Institute of Advanced Technology, Kinki University 10

      Pages: 63-68

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Effect of dimethyl sulfoxide (DMSO) on development of cloned embryos.2005

    • Author(s)
      Kunieda T.
    • Journal Title

      Memoirs of Institute of Advanced Technology, Kinki University 10

      Pages: 53-56

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] ウシ卵子成熟用培地への不飽和脂肪酸の添加が体細胞核移植における細胞融合に及ぼす影響.2004

    • Author(s)
      佐伯和弘
    • Journal Title

      近畿大学先端技術総合研究所紀要 9

      Pages: 9-14

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] ウシ精子の生死が顕微受精卵のMPF活性に及ぼす影響.2004

    • Author(s)
      藤浪菜穂子
    • Journal Title

      近畿大学先端技術総合研究所紀要 9

      Pages: 15-20

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] ゴールデンハムスター顕微授精胚発育に及ぼす遮光及び抗酸化剤の影響.2004

    • Author(s)
      川澄みゆり
    • Journal Title

      近畿大学先端技術総合研究所紀要 9

      Pages: 21-26

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Functional expression of a Δ 12 fatty acid desaturase gene from spinach in transgenic pigs.2004

    • Author(s)
      Saeki K
    • Journal Title

      Proceedings of the National Academy of Sciences of the United States of America 101

      Pages: 6361-6366

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Activation with ethanol improves embryo development of ICSI-derived oocytes by regulation of kinetics of MPF activity.2004

    • Author(s)
      Fujinami N
    • Journal Title

      Journal of Reproduction and Development 50

      Pages: 171-178

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] 体細胞クローンウシの作出および遺伝的同一性・発育相似性の検討.2004

    • Author(s)
      谷口俊仁
    • Journal Title

      日本胚移植学雑誌 26

      Pages: 57-65

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] The effect of addition of unsaturated fatty acid into a maturation medium fo bovine oocytes on the efficiency of electrical fusion of bovine somatic cells an the enucleated oocytes.2004

    • Author(s)
      Saeki K.
    • Journal Title

      Memoirs of Institute of Advanced Technology, Kinki University 9

      Pages: 9-14

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Effects of sperm viability on kinetics of MPF activity of bovine oocytes following ICSI.2004

    • Author(s)
      Fujinami N.
    • Journal Title

      Memoirs of Institute of Advanced Technology, Kinki University 9

      Pages: 15-20

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] The effect of shutting out visual light and anti oxidant on the early development of Intracytoplasmic sperm injection-fertilized eggs.2004

    • Author(s)
      Kawasumi M.
    • Journal Title

      Memoirs of Institute of Advanced Technology, Kinki University 9

      Pages: 21-26

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Functional expression of a Δ12 fatty acid desaturase gene from spinach in transgenic pigs.2004

    • Author(s)
      Saeki K.
    • Journal Title

      Proceedings of the National Academy of Sciences of the United States of America 101

      Pages: 6361-6366

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Activation with ethanol improves embryo development of ICSI-derived oocytes by regulation of kinetics of MPF activity.2004

    • Author(s)
      Fujinami N.
    • Journal Title

      Journal of Reproduction and Development 50

      Pages: 171-178

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Production, genetic identification and similarity of growth degree of cloned cattle developed from embryos reconstructed with fibroblasts from ear tissue of Japanese Black calves.2004

    • Author(s)
      Taniguchi S.
    • Journal Title

      Japanese Journal of Embryo Transfer 26

      Pages: 57-65

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Fertilization by intracytoplasmic sperm injection and subsequentembryo development in vitro to blastocysts in Japanese Monkey.2003

    • Author(s)
      Hosoi Y
    • Journal Title

      Journal of Mammalian Ova Research 20

      Pages: 34-40

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Cryotopによるウサギ未受精卵の凍結保存とその後の受精能力の検討.2003

    • Author(s)
      柴垣美穂
    • Journal Title

      日本受精着床学会雑誌 20

      Pages: 137-138

    • Description
      「研究成果報告書概要(和文)」より
  • [Journal Article] Fertilization by intracytoplasmic sperm injection and subsequent embryo development in vitro to blastocysts in Japanese monkey.2003

    • Author(s)
      Hosoi Y.
    • Journal Title

      Journal of Mammalian Ova Research 20

      Pages: 34-40

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Cryotop vitrification of rabbit oocytes.2003

    • Author(s)
      Shibagaki M
    • Journal Title

      Journal of Fertilization and Implantation 20

      Pages: 137-138

    • Description
      「研究成果報告書概要(欧文)」より
  • [Book] 食べても太らない肉をつくる-植物遺伝子の家畜への導入と機能的発現-、いざ生の扉へ-クローンとエビジェネティクスの新展開-2006

    • Author(s)
      佐伯和弘
    • Total Pages
      18
    • Publisher
      アドスリー
    • Description
      「研究成果報告書概要(和文)」より
  • [Book] Approach to gate of the birth (Production of healthy meat that prevents obesity, -Functional expression of a plant gene in transgenic domestic animals-.)2006

    • Author(s)
      Saeki K
    • Total Pages
      105-122
    • Publisher
      Adthree, Tokyo, Japan
    • Description
      「研究成果報告書概要(欧文)」より

URL: 

Published: 2007-12-13  

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