【摘要】 睡美人(sleeping beauty,SB)是Tc1/mariner转座子超家族的一员,为探索睡美人转座子与不同的转座酶搭配使用及与同种转座酶不同比例搭配使用时的转座效率,本实验构建了SB真核表达重组载体PT2-SV40-EGFP,通过荧光显微镜和实时荧光定量PCR(qRT-PCR)检测进行了猪胎儿成纤维细胞系(PEFs)转染条件优化研究。研究结果表明,不同转座酶与转座子按10∶1混合后细胞转染效率相似,但报告基因表达水平有较大差异,其中SB100X转座酶与转座子混合后细胞的表达水平最高;转座子PT2-SV40-EGFP质粒和SB100X转座酶质粒按不同比例混合转染细胞研究表明,细胞转染效率相似,但表达水平存在显著差异,其中1∶1组最高,约是5∶1组和10∶1组的50倍。本实验优化了睡美人转座子的转座条件为制备转基因动物深入研究提供参考。更多还原

【Abstract】 The sleeping beauty(SB) transposon is a Tc1/mariner family transposon.In order to examine the transposition efficiency of SB when it is used with different transposases and the same transposase at different ratios,our study constructed recombinant vector which was transfected into porcine(Sus scrofa) fetal fibroblasts cells(PEFs) and its transfection efficiency was detected by fluorescence microscope observation and qRT-PRC in an attempt to improve the transfection condition.The results of PT2-SV40-EGFP with different transposase at the same ratio of 10∶1showed that the transfection efficiency of EGFP(enhanced green fluorescent protein) was close,but the difference between expression of EGFP was significant,and SB100X has the highest expression.The results of PT2-SV40-EGFP with SB100X at different ratios showed that expression difference was significant at similar transfection efficiency.The expression level of PT2-SV40-EGFP and SB100X mixed at the ratio of 1∶1 was 50 times as high as the the group at the ratio of 5∶1 and the group at the ratio of 10∶1.This will provide technological platform for producing transgenic animals to further study SB transposon.更多还原