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中文摘要：
利用“睡关人”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10^6～10^7／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。
英文摘要：
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.
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