鸡X期胚盘细胞电转染外源基因条件的探索

以EGFP为报告基因体外电穿孔转染鸡X期胚盘细胞,探讨适宜电转染条件以及影响鸡ES细胞存活率和转染率因素。分离鸡X期胚盘细胞进行培养、传代及鉴定,二代细胞设定不同的电压、脉冲时间、质粒浓度、细胞密度及孵育温度等条件转染。电击后10 min,0.4%台盼蓝染色,血细胞计数板计数,计算存活率。48 h后在荧光显微镜下计绿色荧光细胞数和总细胞数,计算转染率。结果表明:电转染鸡ES细胞的最优化电压为280 v,脉冲时间为75μs,质粒浓度为20μg/mL,转染前4℃、转染后25℃(室温)或4℃各静置10 min,细胞密度调整在1×105个/mL×106个/mL,尽量使细胞处于对数生长期。电穿孔法转染鸡ES细胞是简便有效的方法,利用优化条件转染ES细胞,可以满足进一步实验的要求。

Transfection of EGFP Genes into X Period of Chicken Blastoderm Cells Mediated by Electroporation

To obtain optimum the best conditions of electric transfection and investigate the major influential factors of electransfection efficiency and the survival rate of chicken embryonic stem(ES)cell,as reporter gene,EGFP was electransfected into X period of chicken blastodermic cells in vitro.The chicken blastodermic cells of X period were seperated,cultured,subculture and identified.The 2nd generation cells were transfected in different voltage,pulse time, plasmid concentration,cell density and incubating temperature.After 10 min electric shocked,the cells were dyed with 0.4%trypan blue,and the cell survival rate were counted.After 48 hours,green fluorescent cells and total cells number were calculated by fluorescence microscope,and the cell transfection efficiency was counted.The optimized parameter was achieved under the condition of voltage 280 v,pulse duration 75μs,plasmid concentration 20μg/mL,before transfection 4℃,after transfection 25℃(room-temperature)or 4℃10 min,cell density 1×105 cell/mL~1×106 cell/mL,as far as possible,cells were in logarithmic phase.Electric transfection is a simple and effective way for chicken ES cells. Transfect ES cells were in the optimized parameter can be used to meet the needs for further experiments.