Construction Of Multi-transposon Vector And Transposon Activity Study In Mammal Cells And Embryos

Transposon is the basic unit of chromosomes which can autonomously replicate and shift in whole genome. Transposon is ubiquitous in organism genome and the process of transposon departing form original site and inserting in another gene is called as transposition. Sleeping beauty(SB), piggyBac(PB), Tol2are commonly used in current research and application of transposon. In order to investigate the characteristics of these transposon expression vector containing these3transposable elements was constructed and transposition efficiencies were compared in mammal cell and mice embryonic level providing important reference for the study of transgenic research and gene trapping. This experiment mainly include:1. Transposition sequences of3transposon were obtained by high fidelity PCR and these5sequences were connected with pMD19-T vector respectively. Sequences were identified after DNA sequencing as100%similarity. Transposition sequences were then cloned to pT2-HB vector successively.2. Fluorescent protein GFP sequence was obtained by cutting green fluorescent protein carrier pCAG-GFP with blunt end restriction endonuclease Scal and PsiI and pT3-P/S/T was cut by blunt end endonuclease MscI at the same time. Both sequences were purified by DNA gel extraction kit and connected to construct pT3-PST-CAG-GFP successfully after confirmation. PT3-PST-CAG-GFP was wrapped by polycation PEI to transfect3T3cells and fluorescence was observed by fluorescence microscope after transfection. The results showed that CAG promoter can effectively drive the expression of GFP in cells and transfection efficiency was more than50%. pT3-PST-CAG-GFP vector was transfected with three transposase expression plasmid:SB100X, PBase, Tol2as the following ratio:pT3-PST-CAG-GFP:SB△DD1:1; pT3-PST-CAG-GFP:pCMV-SB100X1:1pT3-PST-CAG-GFP:pCMV-TOL21:1; pT3-PST-CAG-GFP:pCMV-HAhyPBasel: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 more than50%. Transfected cells were collected and extracted genome using cell genomic DNA extraction kit. Relative copies of GFP and cutting efficiency were detected by RT-PCR, single copy gene Amp as internal reference, genome extracted as template.The results suggested that for GFP relative copies, SB group was higher than that of Tol2and PB and experimental groups were higher than the control group, results were analyzed by SPSS and differences between4groups were not significant (P<0.05). For cutting efficiency, SB group was higher than PB group, PB group higher than Tol2group, results were analyzed by SPSS and differences between3groups were not significant (P<0.05).3. pT3-PKG-NEO vector was constructed which can express resistant product allowing cells to survive in G418containing selective medium. NEO gene, approximate1700bp, was amplified by high-fidelity DNA polymerase from plasmid PB-SB-PGK-NEO-bpA and then connected with pMD19-T vector. NEO TA cloning vector was constructed successfully confirmed by DNA sequencing with100%similarity. NEO TA cloning vector and pT3-PST multi-transposon vectors were cut with EcoRI and Xmal restriction endonuclease and then connected with each other. DNA sequencing indicated pT3-PKG-NEO was successfully constructed. pT3-PKG-NEO transfected with3T3cells, the test groups were set as SB, Tol2, PB, 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;SB group was significantly higher than PB and Tol2groups (p<0.05) and experiment groups were extremely significantly higher than control group (p<0.01) but differences between PB and Tol2groups were not significant (p>0.05)4. We injected mouse fertilized eggs though cytoplasm injection method as following solution:pCMV-SB100X:pT3-PST-CAG-GFP1:1; pCMV-HAhyPBase:pT3-PST-CAG-GFP1:1; pCMV-TOL2:pT3-PST-CAG-GFP1:1, each with two replicates, results showed that positive rate of embryos of SB and PB groups were significantly higher than that in Tol2group, the positive rates of SB and PB were close, SB was slightly higher. The highest positive rate of SB, PB, Tol2group was54.67%,48.89%,38.89%respectively.The experiments conducted in this report showed that the multi-transposon vector containing SB、PB、Tol2was successfully constructed; this vector can express GFP and realize transposition in3T3cells effectively, positive embryos were obtained in the experiment of mouse fertilized eggs injection though cytoplasm.The transposition efficiency of SB was higher than PB and Tol2in the level of cell and embryo.