| Project/Area Number |
12556057
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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 | 展開研究 |
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| Research Field |
Applied veterinary science
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| Research Institution | Yamaguchi University |
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Principal Investigator |
SUZUKI Tatsuyuki Yamaguchi University, Faculty of Agriculture, Professor, 農学部, 教授 (00216409)
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| Co-Investigator(Kenkyū-buntansha) |
OTOI Takeshige Yamaguchi University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (30311814)
藤原 昇 九州大学, 農学部, 教授 (60150512)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥8,200,000 (Direct Cost: ¥8,200,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2000: ¥4,700,000 (Direct Cost: ¥4,700,000)
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| Keywords | EGFP gene / bovine / liposome / donor cell / enucleation / reconstructed embryo / transfer / gene expression / EGFPcDNA / 体細胞 / クローン / リポフェクション / 遺伝子導入 / 陰圧式培養器 / クローン子牛の作出 / マイクロインジェクション / 蛍光発現 / エレクトロポーレーション / モザイク / 精子 / 牛初期胚 / ブラストメアー |
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| Research Abstract |
The donor nuclei were prepared from bovine fetuses cell lines, which were derived from frozen-thawed fetus that had been frozen at -20C for about three months. Seven to twelve passages of the cell lines were used as donor nuclei in this study. We conducted to transfer the EGFP gene fragment into the bovine fetus fibroblasts using Liposome. The cells were in D-MEM medium supplemented with 0.5% serum for 4-5d before being used as donor nuclei. The recipient oocytes were prepared from in vitro matured oocytes, which were enucleated at 20-22 h after the onset of in vitro maturation. The enucleation process was done by push out the first polar body and a small amount of cytoplasm after cutting the zona pellucida with a sharp glass needle. All manipulations were done in 20μl drops of m-SOF supplemented with 5μg/ml cytocharasin B and 0.3% BSA covered by mineral oil. The fusion was initiated by a single DC pulse of 1 kv/cm for 50μsec. These reconstructed embryos were cultured during 8 days. The rate of fluorescence expression for whole and mosaic embryos were 11(3.5%) and 26(8.4%), respectively. However, whole fluorescence expression embryos at the blastocysts stage was 3(1%). Whole process for culture of NT embryos, we used the portable CO2 incubator and some reconstructed embryos were carried to China from Japan by air. After reaching to farm of Raiyan Agricultural University, each two NT embryos were transferred to 5 recipients. Finally we obtained 2 NT calves. However, we not confirm the EGFP gene expression from these NT calves.
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