| Transposition is a general biological process, which palys important role in the evolution of species and is one of the major factors determinating the genome size variation. During the lonterm history, transposons display a dramatically different evolution profiles across species, the retrotransposon L1 and ERV are the main active elements in mammals, while the ERV retrotransposons may still active in birds, however both of mammals and birds contain very few active DNA transposons. In contract, fish contain diverse active DNA transposons. Due to the simple structure and the mobility within genome by cut and paste mechanism, some DNA transposons were modified into very useful tools to be applied in genetics research, such as Sleeping Beauty (SB) transposon system. Among them, SB, PB and Tol2 transposons were most widely used, some great progresses have been made in the fields of gene therapy, transgenesis, and functional genetics research. However the current available information on the insertion preference, transposition activity and the technique parameters of intracytoplamic injection mediated with transposons is still very fragmented, and china was very lack of intellectual property of developed transposons. Here we compared the efficiency of integration and gene trapping of these three transposons in cells and embryos, and optimized the technique parameters of intracytoplamic injection mediated with transposons, to facilitate the application of these transposons within animal transgenesis, gene therapy, and functional genomics. At the same time, we try to characterize the highly active DNA transposons by bioinformationic analysys, and set up the intellectual property of transposon system in china. The main results included:1, The transposon plasmid of pT3-PGK-NEO and the transposase plasmids of SB, Tol2, and PB were mixed with different ratio and cotransfected with MEF and HELA cells, to compare the intergration efficiency among three transposon systems. Cells were cultivated in G418 selection medium for 10-15 days to compare positive clones in different groups. The results showed that the number of MEF and HELA positive clones is highest at the ration of 2:1 of transposon and the transposase plasmid for PB and Tol2, which represented the highest intergration efficiency; while the highest number of positive clones of SB, representing the highest intergration efficiency, is at the ration of 1:1 of transposon and the transposase plasmid; at all rations of transposon and the transposase plasmids, the intergration efficiency of SB is higher than that of PB and Tol2; the intergration efficiency of SB at the top ratio is significantly higher than that of PB and Tol2 (p<0.05).2, The transposon plasmid of pT3-PGK-NEO and the transposase plasmids of SB, Tol2, and PB were mixed with different ratio and cotransfected with MEF and HELA cells, to compare the gene trapping efficiency among three transposon systems. Cells were cultivated in G418 selection medium for 10-15d to compare positive clones in different groups. The results showed that the number of MEF and HELA positive clones is highest at the ration of 2:1 of transposon and the transposase plasmid for SB, PB and Tol2, which represented the highest gene trapping efficiency; the gene trapping efficiency of PB is highest and it is significantly higher than that of SB and Tol2 at the top ratio in the HELA cells (p<0.05).3, The transposon plasmid of pT3-PGK-NEO (20ng/ul) and the transposase plasmids of SB, Tol2, and PB were mixed with the different concentrations (lOng/ul,30ng/ul,50ng/ul), respectively, and coinjected into mouse fertilized eggs through cytoplasm injection method. The embryos were cultured to 4 days, and the GFP signal was checked by microscope. The results showed that positive GFP embryos of SB, PB, and Tol2 were above 30%; the highest positive rate of PB was at the concentration of 50ng/ul of transposase mRNA, which is over 50%, and significantly higher than that at the concentrations of 50ng/ul and 30 ng/ul, and also significantly higher than that in SB and Tol2. The highest positive rate of SB and Tol2 was at the concentration of 30ng/ul of transposase, which was 45.00% and 37.16%, respectively. While the GFP positive rate of Tol2 was relatively lower than that of SB and PB.4, By annotating the mobilomes of four representative teleost fishes zebrafish, medaka, stickleback and tetraodon, an unprecedented diversity of active mobilomes within teleost genomes was revealed, and teleost genomes are very active. The differences of mobilome expansions, which dominated by DNA transposons, explain the main size variations of genomes across teleost. Two superfamilies of DNA transposons (hAT and Tel/Mariner) account for a significant portion of the genome size variations present in the teleost genus, and diverse active and intact transposons were found within hAT and Tel/Mariner superfamilies, which indicates that these are active families. The contrasting proliferation histories of retrotransposons were observed across four species as well, and diverse active retrotransposon (DIRS, Gypsy, LI, and L2) was observed in zebrafish and stickleback.6, By bioinformatics analysis, and cell and embryo experimental test, a substantial difference of activity of Tel transposons were found in zebrafish, and the Tcl-c is the youngest, which can transposition effectively in mammal MEF and HELA cells, and efficiently transfer the GFP in mice embryos, and the transposition activity is similar ot that of SB.Overall, this study revealed that, in the celluar level, SB represents the highest intergartion efficiency, while PB represents the highest gene trapping efficiency; Whereas, in the embryoic level, PB represents the highest gene transfer efficiency. The new identified Tcl-c DNA transposon is very active, and has a promising application value. This study also proved that the the technique of intracytoplamic injection mediated with transposons is applicable in animal transgenesis, and we obstained a serious of important research data related with transposons at the celluar and embryoic levels. |
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