Enhancement On Exogenous Gene Integrity And Gene Expression By Sleeping Beauty Transposon System

The Sleeping Beauty (SB) transposon is a member of Tc1/mariner family transposons that has been transpositionally inactive for over 10 million years. The SB transposon system was awakened from inactive Tc1-like transposable elements by using molecular phylogenetic data in 1997. Recent studies on its transposition efficiency and mechanism have shown its broad applications in vertebrate animals for gene-screening, gene transfer, and human gene therapy.In this study, the trangenic efficiency introduced by SB system in mouse and effect on gene immunization of Sleeping Transposase was analysed. Firstly, method of injection was optimized by exlporing the size of injector pinhead, injecting times and hormone in this research. The vector PT2-EGFP was constructed by inserting 8.4kb fragment including EGFP and its 5'and 3'regulated sequence from constructed seminal vesicle vector(PC-EGFP) into PT2-HB. Transgenic animals were obtained by direct testis injection and injection mediated by SB transposon system respectively.The transgenic rate were evaluated and compared between these two methods by detection of the fluorescence of vaginal plug and section of seminal vesicle under fluorescence microscope and PCR. In addtion, the different ratio of tansposon and tansposase in SB transposon system was assessed for high transgenic rate. The results demonstrated that the transgenic rate and litter size of offspring in the groups injected by NO.4 pinhead and injected three times were higher. The transgenic rate mediated by SB(43%) was higher than the transgenic rate by direct testisinjection(20%). However, the transgenic rate was not significant different among the groups with different ratio of transposon and transposase from 5:1-20:1. The results demontrated that the Sleeping Beauty system can enhance the transgenic rate.To investigate the effect of Sleeping Beauty transposase on gene immunazition, the vector pA-hLF was constructed and expressed lactoferritin for gene immunization in Newsland rabbits. 8 weeks old rabbits were divided into four groups: group 1, nake vector-pA-hLF;group 2, PEI-coated pA-hLF;group 3, PEI coated complex with Sleeping Transposase and pA-hLF at a molar ratio of 4:1,;group 4, the control group withough injection.Then intrmuscula immunizations were conducted every two weeks in first 6 week and the final injection was given in 18th week. the blood was sampled and evaluated the titer. After the fourth immunizations the third group produce the antibody against hLF by 1:6400 higher than the first group (1:1600) and the second group (1:2000), and the control group showed no antibody. The result indicate that SB tansposase can promote the expression of exogenous gene, which could be used as a new adjuvant in gene immunization.