牛输卵管上皮细胞转人胶原蛋白cDNA基因及转基因克隆胚胎

以2岁奶牛输卵管为材料，用胰酶消化法分离得到了牛输卵管上皮细胞。输卵管上皮细胞在DMEM／F12培养基中生长良好。用一代牛输卵管上皮细胞为靶细胞，对其进行了电穿孔转染，转染对象是分子大小为31085bp的以β-casein启动子为基础的含有人胶原蛋白cDNA基因和EGFP、Neor双标记基因的质粒。电穿孔试验发现，牛输卵管上皮细胞在低渗缓冲液中电转染可以获得阳性转染子，其中以90mOsm／kg为最佳。电压以800V为好，电压高和低都不利于电穿孔的成功。成功转染的细胞用800mg/L G418进行筛选，在10d后获得了较大的阳性细胞克隆簇。对获得的阳性细胞克隆簇进行扩大后，得到了较纯的阳性细胞克隆系，流式细胞仪检测其纯度为81．6％。在荧光显微镜下选择阳性细胞作为核移植供体细胞进行了转基因克隆试验，结果显示以转基因和非转基因细胞为核供体获得的重构胚融合率差异显著（51．9％比63．2％），获得的桑椹胚／囊胚率差异不显著（20．3％比25．5％）。对获得的具有绿色荧光的胚胎进行DNA分析，结果显示这些胚胎成功转入了外源基因，并且转入的外源基因结构完整。

Bovine oviduct epithelial cells transfected with human collagen cDNA gene and transgenic cloning embryo

Isolated bovine oviduct epithelial ceils by trypsin digestion from oviduct tissue of a 2-year-old dairy cow. Oviduct epithelial cells grow well in DMEM/F12 medium. The first generation oviduct epithelial cells were used as target cells to carry out electroporation transfection. Molecular size of the transfected genes is 31 085 bp. The gene is a plasmid that carry β-casein promoter,human collagen eDNA and EGFP,Neor as double marker. Electroporatioa experiment found that bovine oviduct epithelial cells can get positive-transfectant in hypotonic buffer. Among them,90 mOsm/kg is the best. For voltage,800 V is the best. High and low voltage are not conducive to the success of electroporation. Successfully transfected cells were screened with 800ug/ml of G418. on day 10, a larger cluster of positive cell clones were obtained. The cluster of positive clone cells were expanded to obtain a more pure cell line. Detected by flow cytomerty,the purity was 81.6% . This cell as a nuclear transfer donor carried out gene transfer experiments. The results showed that in transgenic cells and nontransgenic cells, their fusion rate of reconstructed embryos obtained significant difference（51.9 % vs 63.2 %）, their morula/blastocyst rate of reconstructed embrys were not significantly different （20.3 % vs 25.5% ）. DNA of the embryos displaying green fluorescence was analyzed,The results showed that the embryos was successfully transferred to the foreign gene and the genetic structure of the transfer was complete.