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Production of human blood cohesion factor from bovine milk by nuclear transfer following the EGFP gene

通过 EGFP 基因核移植从牛乳中生产人血凝聚因子

基本信息

批准号：
12556057
负责人：
SUZUKI Tatsuyuki
金额：
$ 5.25万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

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.

供体核来自牛胎儿细胞系，这些细胞系来自冷冻解冻的胎儿，这些胎儿在-20℃下冷冻了大约三个月。本研究选用7~12代细胞作为供核。我们利用脂质体将绿色荧光蛋白基因片段导入牛胎儿成纤维细胞。细胞在添加0.5%血清的D-MEM培养液中培养4~5d后作为供核。受体卵母细胞取自体外成熟的卵母细胞，于体外成熟后20~22h去核。用锐利的玻璃针切断透明带后，通过推出第一极体和少量细胞质来完成去核过程。所有操作均在20μL滴加5μg/ml细胞粘附素B和0.3%牛血清白蛋白覆盖矿物油中进行。融合由1千伏/厘米的单一直流脉冲启动，持续50μ秒。这些重组胚胎在8天内进行了培养。完整胚胎和镶嵌胚胎的荧光表达率分别为11个(3.5%)和26个(8.4%)。而囊胚期的完整荧光表达胚胎为3枚(1%)。在NT胚胎培养的整个过程中，我们使用便携式CO_2孵化器，将部分重构胚胎从日本空运到中国。到达莱阳农业大学农场后，将每2个NT胚胎移植给5个受体。最终我们得到了2头NT小牛。然而，我们还没有证实这些NT牛犊中是否有EGFP基因的表达。