摘要
[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。
[ 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 caiTied out amplification culture by con-culture, neo gene was as screened gene, genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [ Result] Compared with non-conditioned culture medium, 100% conditioned culture medium could greatly increase survived rate of single colony cells. Compared with control, con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification cuhure （FF： 53.33% vs. 10.00% ;MGE： 33.33% vs. 6.67% ）, confluence time of amplification culture was significantly decreased （20 - 30 d）. The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [ Conclusion] The study provides a reliable method for separating transgenic cell, inserting and diagnosing ideal vector, and can save expense and time for transgenic animal production.
出处
《安徽农业科学》
CAS
北大核心
2009年第31期15146-15149,共4页
Journal of Anhui Agricultural Sciences
基金
河南科技大学博士启动基金项目
关键词
细胞
单克隆
扩大培养
转基因
山羊
Cell
Single colony
Amplification culture
Transgene
Goat
