山羊转人乳铁蛋白基因成纤维细胞和乳腺上皮细胞单克隆的制备及扩大培养

目的]为探索单个转基因阳性细胞快速扩大培养成为细胞克隆的技术体系。方法]对转染人乳铁蛋白(Human lactoferrin,hLF)基因乳腺特异性表达载体pBLM-C1的单个山羊胎儿成纤维细胞(Fetal Fibroblasts,FF)和乳腺上皮细胞(Mammary Gland Epithelial,MGE)细胞进行克隆。在96孔板中,首先用3种浓度(V/V:0、50%和100%)的适应性条件培养基对单个转染细胞进行细胞单克隆的制备,进而把转染细胞单克隆与非转染细胞共培养进行扩大培养,neo基因被用于筛选基因,以PCR方法鉴定转染细胞基因组DNA。并对单克隆细胞进行染色体核型分析。结果]与非适应性条件培养基相比,100%适应性条件培养基能够显著提高细胞单克隆存活率;与对照相比,转染细胞单克隆与非转染细胞共培养,显著提高了转染细胞单克隆扩大培养后的比率(FF:53.33%vs.10.00%;MGE:33.33%vs.6.670%),且明显缩短了扩大培养汇合时间(20～30 d);PCR鉴定结果表明,上述方法获得的克隆细胞整合有hLF目的基因;核型分析表明,大部分细胞克隆染色体正常。结论]该研究可为分离转基因细胞、理想载体的插入及诊断提供一种可靠的方法,并能节省转基因动物生产的及费用时间。

Single Colony Preparation and Amplification Culture of Fetal Fibroblast and Mammary Gland Epithelial Cell of Human Lactoferrin Transgene from Goat

Objective] The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture.Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned.Single cell colony of single transfection cell was prepared with 3 concentrations of 0,50% and 100% conditioned culture media.Transfection cell and non-transfection cell were carried out amplification culture by con-cul...