| Project/Area Number |
15380196
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied animal science
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| Research Institution | Kinki University |
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Principal Investigator |
SAEKI Kazuhiro Kinki University, School of Biology-Oriented Science and Technology, Professor, 生物理工学部, 教授 (10298937)
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| 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)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥7,800,000 (Direct Cost: ¥7,800,000)
Fiscal Year 2005: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2004: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | α-linolenic acid / ω3 fatty acid desaturase / bovine fibroblast cells / mouse 3T3-L1 cells / codon usage / luciferase expression vector / cloned embryos / DNA methylation / ω3脂肪酸不飽和化酵素遺伝子 / ウシ初代繊維芽細胞 / stableな遺伝子導入 / 初期G1期細胞 / リポーター遺伝子発現 / Ki-67タンパク質 |
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| 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
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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
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