睡美人转座子真核表达载体的构建及在猪胎儿成纤维细胞系中的表达验证  

Construction of Sleeping Beauty(SB) Transposon Vector and Its Expression in Porcine(Sus scrofa) Fetal Fibroblasts Cells

作　　者：周家庆[1,2] 王升智[1] 宋成义[1,2] 高波[1] 李奎[2] 钱丽丽[2] 崔文涛[2] 牟玉莲[2] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院/国家动物遗传育种与繁殖重点(培育)学科,扬州225009 [2]中国农业科学院北京畜牧兽医研究所/农业部畜禽遗传资源与利用重点开放实验室,北京100094

出　　处：《农业生物技术学报》2013年第6期684-689,共6页Journal of Agricultural Biotechnology

基　　金：中国博士后科学基金特别资助项目(No.201104168);国家自然科学基金(No.31200920);国家转基因新品种培育重大专项(No.2011ZX08011-006)

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。The sleeping beauty （SB） transposon is a Tcl/mariner family transposon. In order to examine the transposition efficiency of SB when it is used with different transposases and the same transposase at differentratios, 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 observationand qRT-PRC in an attempt to improve the transfection condition. The results of PT2-SV40-EGFP with different transposase at the same ratio of 10：1 showed that the transfection efficiency of EGFP（enhanced greenfluorescent 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 furtherstudy SB transposon.

关 键 词：睡美人(sleeping beauty SB) 真核表达载体 转基因动物 

分 类 号：S433.5[农业科学—农业昆虫与害虫防治]