“睡美人”转座子系统介导EGFP转染鸡SSCs条件优化的研究  

Optimizing Conditions of Transfecting Chicken SSCs with EGFP by the Sleeping Beauty Transposon

作　　者：李碧春[1] 陈强[1] 高波[1] 田智泉[1] 余飞[1] 何先红[1] 宋成义[1] 常国斌[1] 王克华[2] 

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009 [2]江苏省家禽科学研究所,江苏扬州225003

出　　处：《中国家禽》2009年第1期10-14,共5页China Poultry

基　　金：国家自然科学基金(30671509,30871791);高等学校博士学科点专项科研基金资助项目(20061117004);国家高新技术863项目(2007AA100504);江苏省自然科学基金(BK2007077);江苏省高校自然科学重大基础研究(08KJA230001)

摘　　要：利用"睡美人"转座子系统介导增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)报告基因,比较和优化了电穿孔转染条件和精原干细胞(spermatogonial stem cells,SSCs)对G418的耐受能力。结果表明,在SB与PT-EGFP的比为1∶10,质粒浓度为15μg/mL,细胞处于对数生长期,且细胞密度为106～107/mL时,精原干细胞在电场强度为270V,脉冲时程为80μs,转染后在4℃静置10min的条件下转染效率最高。浓度为400μg/mL的G418对转染后的SSCs进行快速筛选6d时,出现了EGFP阳性克隆。In this study,enhanced green fluorescent protein gene （EGFP）as marker gene was electroporately transfected into chicken spermatogonial stem cells by the Sleeping Beauty（SB）transposon to compare and emphasize the optimization of electroporation parameters. We also focused on the proper concentration of G418 through cell toxicity sensitivity experiments to fastly select the transfeeted SSCs in vitro. The results showed that primary generation spermatogonial stem cells（SSCs）had a highest fluorescent expression rate （24.25%）when the rate of the SB and PT-EGFP was 1：10. The optimization electroporation parameters were the 270 V,80 μs pulse,15 μg/mL plasmid concentration,10^6 to 1×10^7 per mL logarithmic growth phase cell placed in 4 ℃ after electroporate transfected. When the fast selection for SSCs after transfection for 6 d at the concentration of 400 μg/mL G418 medium,the SSCs positive clones had been found.

关 键 词：转座子 电穿孔 精原干细胞 增强型绿色荧光蛋白 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]