Construction Of Gene-trap Vector Mediated By Multi-transposon And Comparison Of Trap Efficiency

Gene trapping techniques, which is one of the current applications of gene cloning methods. Use the technology to build a random insertion mutant library, can be used to find, identify and research a large number of unknown and activation of genes of known function. Transposon is the basic unit of chromosomes which can autonomously replicate and shift in whole genome. This study combines the technique of gene trap and transposon, gene trap vector containing these3transposable elements was constructed and transposition efficiencies were compared in mammal cell and zebrafish embryonic level providing important reference for the study of transgenic research and gene trapping. This experiment mainly include:1. Gene splicing receptor (SA) components was cloned by high-fidelity PCR method from human genome and sequences were identified after DNA sequencing as100%similarity. Connect this fragment with pIRES-GFP carrier, PCR amplification of containing S A-IRES-GFP-SV40polyA four components, Sequences were identified after DNA sequencing as100%similarity. The fragment inserted transposon vector pT3-PST built into the promoter trap vector pT3-PST-Pro-Trap. pT3-PST-Pro-Trap vector was transfected with three transposase expression plasmids:SB100X, PBase, Tol2as the following ratio:pT3-PST-CAG-GFP:SB△DD1:1; pT3-PST-CAG-GFP:pCMV-SB100X1:1;pT3-PST-CAG-GFP:pCMV-Tol21:1;pT3-PST-CAG-G FP:pCMV-HAhyPBase1:1,a negative control group was set using inactive transposase vector: SB△DD. There were3replicates of each group, green fluorescent protein expression level was observed48h after transfection. The results showed that CAG promoter can effectively drive the expression of GFP in cells and transfection efficiency was high that the control group. The green fluorescent protein expression level of PB are more that SB and Tol2.2. pT3-NEO-Trap vector was constructed which can express resistant product allowing cells to survive in G418containing selective medium. NEO gene was amplified by high-fidelity DNA polymerase from plasmid PB-SB-PGK-NEO-bpA and the vector was constructed successfully confirmed by DNA sequencing with100%similarity. NEO TA cloning vector and pcDNA3vectors were cut with EcoRI and KpnI restriction endonuclease and then connected with each other. The vector was called pcDNA3-NEO-bpA.Then SA and pcDNA3-NEO-bpA were cut with XmaI and BamHI restriction endonuclease and then connected with each other. The vector was called pcDNA3-SA-NEO-bpA. Finely, pcDNA3-SA-NEO-bpA and pT3-PST were cut with XmaI and EcoRI restriction endonuclease and then connected with each other. DNA sequencing indicated pT3-NEO-Trap was successfully constructed. pT3-NEO-Trap transfected with MEF andHELAcells, the test groups were set as SB, Tol2, PB, accordance with50:1、10:1、2:1、1:1、1:2, negative control group was SB△DD. Cells were cultivated in G418selection medium for5d to compare positive clones in different groups. The results showed that positive clones in the experimental group were significantly higher than that in control group; PB group was significantly higher than SB and Tol2groups (P<0.05) and experiment groups were extremely significantly higher than control group (P<0.01),Transposons and transposase2:1captured at the highest efficiency.3.The vector plasmid DNA pT3-PST-Pro-Trap and transposase mRNA of SB, PB and ToL2according to1:1.5ratio was mixed, respectively and injected into zebrafish (Danio rerio) one-cell embryos. At the same time for an injection embryos as the control group. Each group of repeat three times, each time injection embryo number average in more than30pieces.The gene trap efficiency could be determined by the expression level of report gene green fluorescent protein (GFP). The results showed that promoter trap vector mediated by SB, PB, and Tol2transposon could capture the endogenous gene promoter with high efficiency of24.32%、30.7%and18.87%36h after injected. PB-mediated gene trap efficiency was significantly higher than that of SB and Tol2(P<0.05). Three kinds of transposon capture efficiency in60h period were higher than36h period, SB and Tol2group were significant differences (P<0.05), while PB group were no significant difference (P>0.05). The results show that PB, SB and Tol2transposon mediated gene capture technology can be used to vertebrate high-throughput screening functional genes, and provide effective new method for the research of functional genes.4. In order to compare the gene trapping efficiency of DNA transposase and mRNA transposase, We use PGBT-RP2-1plasmid DNA and Tol2transposase DNA, mRNA, respectively at a mass ratio of1:1.5injection of one-cell embryos of zebra fish, while not embryos for injection as control group. At least three of each set of duplicates, number of injected embryos at once over30.After injection, respectively36h and60h expression in fluorescence under a fluorescence microscope, and recorded by the two embryo survival rates, the positive rate in the period. Results showed that in To12transposon vector pGBT-RP2-1under enzyme DNA, mRNA gene trapping, fluorescent expression occurs.36h To12-mRNA about the fluorescence detection of capture efficiency of the capture efficiency is higher than CMV-Tol2(P<0.05);60h detection more efficient than fluorescent expression36h, the capture efficiency of the capture efficiency is higher than CMV-To12and Tol2-mRNA (P<0.05).The experiments conducted in this report showed that the multi-transposon gene trap vector containing SB、PB、Tol2was successfully constructed; The gene trap vector can efficiently capture gene in mammalian cells and zebrafish embryos. The results indicate that PB transposon system in the level of cells and embryonic genes capture efficiency is better than that of SB and Tol2. Transposase in the form of mRNA can significantly improve the efficiency of gene trap. This research for the higher animals functional gene research methods provide effective reference.