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 moth Trichoplusia 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 of GFP, detection of GFP expression was done by fluorescence microscopy, and real-time fluorescent quantitative PCR, after cell genome extraction and using Amp 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). The GFP 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.

Key words: Sleeping beauty, piggyBac, Tol2, embryo fibroblast, transposon activity