重组人gdnf乳腺特异表达载体转染牛胎儿成纤维细胞研析.pdfVIP

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN 年第 期 2014 8 重组人 gdnf 乳腺特异表达载体转染牛胎儿 成纤维细胞的研究 虞飞  李斌  张晶晶  丁海麦  张学明 （内蒙古科技大学包头医学院，包头 014040） 摘 要 ： 为了制备重组人GDNF 牛乳腺生物反应器，采用组织块贴壁法分离培养雌性牛胎儿成纤维细胞，连续继代培养75 d，进行形态观察和染色体分析，在此基础上，转染带有新霉素抗性和红色荧光蛋白双重筛选标记的重组人gdnf 乳腺特异表达载 体pNR-GDNF，G418 筛选阳性抗性克隆，进行PCR 法鉴定。结果表明，分离培养的牛胎儿成纤维细胞具有正常的形态、分裂增殖 特性和染色体数目 ；目的基因已整合到转基因细胞的染色体上。 关键词 ： 牛胎儿成纤维细胞 分离培养 转基因 GDNF Transfection of Bovine Fetal Fibroblast with the Vector for Expressing Human GDNF Specifically in Mammary Gland Yu Fei Li Bin Zhang Jingjing Ding Haimai Zhang Xueming （Baotou Medical College ，Inner Mongolia University of Science and Technology ，Baotou 014040 ） Abstract:  In order to prepare bovine mammary gland bioreactor for production of human recombinant GDNF, the female bovine fetal fibroblast cells were successfully isolated using tissue bulk attachment. The fibroblast cells were cultured consecutively for 75 days for further morphologic observation and chromosome analyzing. The plasmid vector pNR-GDNF, which contained the Neor gene and the DsRed2 gene as positive selection marker genes and human GDNF cDNA gene regulated by bovine beta-casein promoter for specific expression in mammary gland, was transfected into the bovine fetal fibroblast cells by electroporation. After selection with G418 for 7 days, resistant cells expressing red fluorescence protein were isolated, cultured, expanded and cryopreserved by standard procedures. The transgenic cells were indentified by PCR. The results showed that bovine fetal fibroblast cells possessed normal morphology, multiplication characteristics and