Optimization Of Enhancer Trapping System Mediated By The Transposon

Enhancer trapping technology,an important method for studying functional genomes,is effective to identify enhancers,mine unknown enhancers in the genome and explore their characteristics on the spatio-temporal expression regulatory through ehancer trapping library.DNA transposon is a gene capable of moving in genome,which is successfully used as a nonvirus-vector for gene transfer.The trapping efficiency has been improved significantly by transposon-mediated enhancer trapping technology,which has been widely used in model organisms such as zebrafish,mice,and fruit flies,but some technical details still need to be improved.In this study,we systematacially optimize enhancer trapping and dectecting vectors focusing on minimal promoters and construct a bidirectional enhancer trapping vector based on zebrafish and mammalian cells.We also established effective methods to trap and identify enhancers in vivo and in vitro,providing a reference method for studying the regulatory characteristics of enhancers.The results mainly include the followings:1.In order to obtain an efficient basic promoters for enhancer trapping and verification,four minimal promoters(Myc,Oct4,krt4 and Gata)were cloned to drive the reporter gene mCherry.The resulted trapping cassettes were inserted into Tol2 transposon vecotr to construct enhancer trapping vectors harboured by Tol2 terminal inverse repeats(TIRs).The enhancer trapping efficiency was assayed by vector plasmids and Tol2 transposase mRNA co-injection into zebrafish zygote.The results showed the enhancer trapping efficiency of Krt4 and Gata group are much higher than that of Myc and Oct4 group.Then two connected chicken HS4 insulators were cloned and inserted into the downstream and upstream of the enhancer trapping boxes of Krt4 and Gata to result two enhancer detection vectors,which were evaluated by zebrafish zygote injection with vectors and Tol2 transposase mRNA.The percentage of 72 hpf embryos showing background fluorescence in Krt4 promoter(about 67%,n=302)is much higher than that in Gata promoter(about 5%,n=301).The results indicate that minimal promotor Gata is good to use for enhancer trapping and detection vector in this study.2.In order to evaluate the regulatory effect of basic promotors on enhancers,another enhancer detection vector was constructed based on the above detection vector by replacing Gata and mcherry with beta-globin and GFP.Then four conservative enhancers(Z48,hand2,hs769,and CNS1)were cloned and inserted into the upstream of Gata and beta-globin btween insulators.The results of zebrafish zygote injection assay indicated that enhancer activity and expression regulatory patterns of Z48 and CNS1 can be verified effectively by Gata,that of Z48,hand2 and CNS1 can be presented effectively through beta-globin.These suggest the interaction effect of enhancers and promoters is significantly affected by difference of promoters.We can evaluate an enhancer by multiple minimal promoters.3.In order to obtain an optimal basic promotor for enhancer trapping and verification technology in mammals,hs278 enhancer were cloned and linked with each of minimal promoters Myc,Oct4,beta-globin,and TATA,then the cassettes were inserted into the MCS of pGL3-basic vector expressing Dual-Luciferase gene through regular molecular biology experimental methods.The resulted vectors were transfected into MEF,C2C12,and 3T3 L1 cell lines to check enhancer activity of hs278.The results of Luciferase assay showed that hs278 enhanced the expression level of Oct4 and TATA in MEF cells,and that of Myc,Oct4 and TATA in C2C12 cells,and that of Myc,Oct4 and beta-globin in 3T3 L1 cells.The results indicate that the constructed enhancer detection vectors can successfully evaluate mammalian enhancers in vitro.Promoter and cell specificity for enhancers were furtherly confirmed.4.In order to furtherly improve the efficiency of enhancer trapping and annotation in zebrafish and mouse,we constructed a bidirectional enhancer trapping vector which mainly included two sets of minimal promoter expression boxes(Gata-mCherry and Gata-GFP)separated by two insulators,splicing acceptor(SA)linked with polyA,PS transposon TIRs carrying the whole trapping cassette.So the bidirectional vector can capture enhancers in the upstream and downstream of the insertion sites at the same time.The results of zebrafish zygote injection with the mixture of the vector and PS transposase mRNA showed that there were no positive embryos expressing mCherry at 12hpf,24hpf and 72hpf stage,while the positive rates of embryos expressing GFP reached 30.2%,61.4%and 36.2%,respectively.The results indicate that the bidirectional enhancer trapping vector can effectively integrate into the genome and capture enhancers,and SA-polyA in the opposite direction helps it function.In summary,we systematically optimize the current enhancer trapping and detection technology.We successfully construct highly effective enhancer trapping and detection vectors for zebrafish and mammals by the combination of cis elements,poly(A),SA,insulator and transposon.We establish a method for enhancer activity assay in vitro and in vivo,reveal the interaction effect between promotors and enhancers,and confirm the specificity of enhancers regulating genes expression.These lay the foundation for further research on the regulatory mechanism of enhancers.