| Transposon is widely distributed in biological universe, and also is the basic DNA sequence of chromosomes which can replicate and shift in whole genome. Transposons can effective mediated gene transfer, and proved to have high application value in gene transfer and gene trapping. Till now, plenty of highly effective DNA transposons are found and separated from biosome, such as Sleeping beauty (SB), piggyBac (PB), To12 et al. These have widely applied in some modle animals gene transfer and gene trapping including zebrafish, mice and eelworm, nevertheless, there still lack of knowledge on transposition characteristics of PB, SB and To12 transposons. In this study, we investigate the gene transfer, gene and enhancer trapping efficiency of three transposons, they are Sleeping beauty, piggybac,and To12. These researches would supply an important reference to transposons related research fields. This experiment mainly including:1.To compare the transfer efficiency of PB,SB and To12 transposons, the GFP expression box of FAG-GFP, containing the beta-actin promoter came from carp (named FAG) coupled with GFP were cloned to transposon structural frame, and obtained 3 transposon-mediated transgenic vectors, and they were pPB-FAG-GFP, pSB-FAG-GFP and pTo12-FAG-GFP. Co-injected with transposase mRNA in five ratios:20ng:10ng,20ng:30ng,20ng:50ng,20ng:80ng,20ng:100ng. The best injection ratio of SB and PB were 20ng:50ng, however, To12 was 20ng:30ng. In gene transfer experiment, FO zebrafish embryos GFP expression ratio were detected at 48hpf and 120hpf■ which were 68% and 68% (N=872) of To12 group, respectively, and higher than PB group (66% and 68%,N=885) and SB (64% and 65%, N=897), but the differences were not significant (P>0.05). According to screening results of F1 zebrafish from FO mating with wild type fish, the germline transmission To12 group was the highest and reached to 46.07% (NF0=231), which was almost 3 folds higher than the 17.86%(NF0=212) in PB group and 13.14% (NF0=199) in SB group.2. To compare the enhancer trapping efficiency of PB,SB and To12 transposons,the GFP trapping box of krt4-GFP,which contained the mini-promoter krt4 came from Danio rerio coupled with GFP were cloned to 3 transposons structural frame, and obtained 3 enhancer trapping vectors: pPB-krt4-GFP, pSB-krt4-GFP and pTol2-krt4-GFP. Co-injected with transposase mRNA in best injection ratio of each transposon. In enhancer trapping experiment:the FO zebrafish embryos GFP expression,were detected at 48hpf and 120hpf, and were 90% and 93% (N=1524) of To12 respectively, which were significantly higher than PB (83% and 83% , N=976) in two observed time points, and a little higher than SB at 48hpf (88%,N=1025), moreover, the differences are significant at 120hpf(89%,N=1025) (P＜0.05). According to screening results of F1 zebrafish from FO mated with wild type zebrafish, the germline transmission To12 group was the highest and reached 55.56% (NFo=165), which was higher than 32.56%( NFo=149) of PB group and 38.36% (NFo=151) of SB group. Screening the patterns of F1 generated from positive funders, the To12 group tended to produce single expression pattern in positive offsprings, while the PB group produced diverse expression patterns offsprings, moreover, SB group performanced between them.3. To investigate the impact of IRES element on gene trapping, the IRES element was inserted into pTol2-RG-Trap downstream of PolyA trapping, to form a new vector of pTo12-GTrap. Co-injected with To12 transposase mRNA in the ratio of 20ng:30ng, and the FO zebrafish embryos GFP were detected at 48hpf and 120hpf, the GFP positive embryos of pTo12-GTrap were 74.93% and 73.91% (N=986), respectively, which were significantly higher than pTo12-RG-Trap group with 41.86% and 41.04% (N=853) (P<0.05).To sum up, the IRES element increased the gene trapping efficiency, and wa applicable in the gene trapping study.4. To compare the gene trapping efficiency of PB,SB and To12 transposons,on the basis of the gene-trap vector pTol2-GTrap, replacing the To12 backbones with PB and SB,to form the pPB-GTrap and pSB-GTrap vectors respectively. Co-injected with the transposase mRNAs and transposon plasmids in best injection ratio for three transposons respectively, and the FO zebrafish embryos GFP expression ratios were detected at 48hpf and 120hpf, the GFP positive embryos in To12 group are 74.93% and 73.91%(N=986) respectively, which are significantly higher than SB (66.07% and 64.02%, N=1025) and PB (54.23% and 53.26%, N=976) in the same observed time, and the differences between 3 transposons were significant in each time tested(P<0.05).Over, the study indicated that To12 transposon has the highest efficiency of these 3 transposons in gene transfer, enhacer trapping, gene trapping and germline transfer. What’s more, IRES was a valid element which could increase the transposon mediated gene trapping efficiencyTo12 transposon exhibit highest gene transfer efficiency. In enhancer trapping study, the To12 tended to produce more positive F1 offsprings, while the PB group produced diverse embryo expression patterns, which could have high application value in enhancer trapping research. |
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