中国农业科学Issue(11)：2270-2278,9.DOI:10.3864/j.issn.0578-1752.2015.11.018
多转座子载体的构建及转座特性比较研究
Construction of Multi-Transposon Vectors, and Comparative Study of Transposon Characteristics
摘要
Abstract
[Objective] Transposons are the basic unit of chromosomes which can autonomously replicate and shift within the whole genome. Sleeping beauty(SB), piggyBac(PB) and Tol2, found from salmonid, cabbage looper mothTrichoplusia ni and Oryzias latipes, respectively, are most active transposons in the vertebrates today. This paper compares the transfection, insertion and cutting efficiencies of the 3 different transposons in 3T3 cells, then obtained the best transposon system at the cellular level.[Method] The 3′ and 5′ terminal transposable elements were cloned from SB, PB and Tol2 transposon vectors using the high-fidelity PCR in this experiment, ensured the accuracy through sequencing, then the transposable elements were subcloned one-by-one into the pT2-HB carrier frame, thus constructed the multi-transposon vectors, pT3-PST, which included all 3 transposons. Green fluorescent protein expression cassette (pCAG-GFP) and neomycin expression cassette (NEO) genes were cloned into pT3-PST carrier, resulting in two expression vectors, pT3-PST-CAG-GFP and pT3-PGK-NEO. These two expression vectors, with the transposase expression plasmid pCMV-SB100X,were mixed with pCMV-HAhyPBase and pCMV-Tol2 at a mass ratio of 1:1, respectively. They were then wrapped by polycation PEI, and co-transfected into the mouse embrynic fibrilasts (3T3). At the same time, a negative control group was set up using the inactive transposase vector SB△DD. After 36 h post-transfection ofGFP, detection ofGFP expression was done by fluorescence microscopy, and real-time fluorescent quantitative PCR, after cell genome extraction and usingAmp as an internal reference. Primers were designed according to the upstream and downstream sequences, then the cutting efficiency of the 3 transposons was determined by real-time fluorescent quantitative PCR. After 48 h post-transfection of NEO, cells were filtered by using 500 µg·mL-1 G418 resistance screening until the normal cells were almost dead (10 d). The cells were stained with Giemsa stain, and the number of drug-resistant cells was counted, and finally different transposon activities of different groups were compared.[Result]Multi-transposon vectors, PT3-PST, pT3-PST-CAG-GFP,and pT3-PGK-NEO were successfully constructed. The transfection efficiency of cells in the experimental groups was more than 50%, and the difference was not significant (P>0.05). TheGFP relative copy number of SB group was higher than Tol2 and PB groups, but the difference was not significant (P>0.05), though it was significantly higher than the control group (P<0.05). The cutting efficiency of SB group and PB groups was significantly higher than Tol2 group(P<0.05), though SB and PB group had no significant difference (P>0.05). pT3-PGK-NEO and transposase co-transfected 3T3 cells, and the results of G418 selection showed that the number of drug-resistant cells of SB group was significantly higher than PB and Tol2 groups (P<0.05), but the difference between PB and Tol2 groups was not significant (P>0.05). However, all the three experiment groups were significantly higher than that of the control group (P<0.01).[Conclusion]The SB transposon system efficiency, at the cellular level, was better than PB and Tol2, which could provide an effective tool for cellular transgenetic research.关键词
SB转座子/PB转座子/Tol2转座子/胚胎成纤维细胞/转座活性Key words
Sleeping beauty/piggyBac/Tol2/embryo fibroblast/transposon activity引用本文复制引用
沈丹,崔恒宓,宋成义,谢雨琇,李庆平,薛松磊,史云强,王赛赛,陈才,钱跃,高波..多转座子载体的构建及转座特性比较研究[J].中国农业科学,2015,(11):2270-2278,9.基金项目
国家自然科学基金（31200920）、国家转基因生物新品种培育科技重大专项（2013ZX08006-005）、中国博士后科学基金特别资助项目（201104168）、江苏省产学研联合创新资金项目 （）
