The Comparison Of The Transposon Activity Among SB, PB And Tol2Transposons In PEF And MEF

Sleeping beauty (SB) piggyBack(PB) and Tol2are different kind of transposons,and he activity of these transposons having difference. In order to compare the activity in PEF and MEF, in this experiment, we constructed the Eukaryotic expression vectors PT2-SV40-eGFP and pT3-PST-CAG-GFP to analyses the transposon activity to optimize experimental condition. Our experiments mainly consist of the following aspects:Firstly, eGFP was cloned and to make sure the sequences are correct through sequencing, then connect to pGL3-SV40, then was connected to pT2-HB to construct pT2-SV40-EGFP. Secondly, pT2-SV40-EGFP was transfected into the PEF and to observe the expression of eGFP48hours later. The result showed that the ration of transfection was high (>50%). This means that Lipo-fectamineTM2000is a effective media of transfection.PB and Tol2were cloned to pT2-HB, in order to construct pT3-PST, then using incision enzyme to obtain the sequences of CAG-GFP, then was cloned to pT3-PST to construct pT3-PST-CAG-GFP which was transfected in the PEF and MEF as the method above.The result showed that pT3-PST-CAG-GFP can express in PEF and MEF, which means the construction of pT3-PST-CAG-GFP is successful. In order to compare the activity of SB11,SB16and SB100x, pT3-PST-CAG-GFP and transposase are transfected into PEF and MEF at the ratio of10:1, then compare the transposon activity through RT-QPCR.The result showed that transposon activity of SB1OOx is higher than that of SB11and SB16.In order to compare transposon activity of SB, PB and Tol2. pT3-PST-CAG-GFP is transfected into PEF and MEF with SB100x, PBase and Tol2TBase respectively at different ratio of10:1,5:1,1:1,1:5and1:10。To observe the expression of GFP through fluorescence microscope and compare transposon activity through the RT-QPCR.The result shows that transposon activity of SB100x is higher than that of PB and Tol2and SB100x, PB and Tol2have different ratio with transposase when they have the highest transposon activity. SB, PB have the same ratio in PEF and MEF SB is1:1, PB is1:10, Tol2has different ratio in PEF and MEF1:10in PEF and1:5in MEF.All of the results above showed that SB, PB, and Tol2can improve the transposon activity PEF and MEF; the activity of SB is higher than PB and Tol2; SB has the Excessive suppression, while PB and Tol2do not have Excessive suppression.