Discovery And Validation Of Tcl/mariner Transposon,Highly Active Members And Their Application In Enhancer Trapping

With the increase of the number of genomes sequenced,the knowledge about the importance of transposons in life science is ongoing increasing.Accumulated studies domestrated that transposons are widely distributed in the organisms,play an important role in the evolution of the host genome,and may impact on the speciation and breed(line)differentiation.In addition,due to the characteristics of transposition,some DNA transposons have been successfully developed into genetic manipulation tools,such as sleeping beauty(SB),piggyBac(PB)and Tol2,which have been widely used in reaearch fields of transgenesis,gene therapy and functional genomics with substantial progresses achieved.Among them,Tc1/mariner is a widely distributed DNA transposon superfamily,and so far,more than ten Tc1/mariner transposons have been identified as active elements in native form,but only SB transposon,which was molecularly reconstructed,have been widely used.Here,we systematically excavated the Tc1/mariner transposons in the zebrafish genome and evaluated the application potential for the high active member,and systematically investigated the evolution profiles of the DD41D(named as Visitor,VS)family,which also belongs to the Tc1/mariner superfamily.The current study included the following contents:1.We performed de novo detection of Tc1/mariner transposons in the zebrafish genome by using multiple pipelines.We carried out phylogenetic classification,insertion age analysis,and domain prediction of transposase for these transposons.2.We investigated the taxonomic distribution,evolutionary relationship,insertion age,and horizontal transfer DD41D/VS transposons,and validated the transposition activity of bat intact VSs in mammal cells.2.We investigated the transposition activity and characteristics for ZB transposon system in mammal cells.3.We applied the ZB transposition system for enhancer trapping in zebrafish and characterized the trapped enhancers.4.We developed the sperm mutant library mediated with ZB transposon system in mice for enhancer trapping study.The main results were as follows:1.The evolutionary analysis of the DD41D/VS family in the Tc1/mariner superfamily showed that VS transposons were widely distributed in the animal kingdom,where 140 invertebrate species,and 30 vertebrates(of which were 12 mammals,including 1 Primates)species,and 1 plant species contain VS elements.VSs have experienced multiple waves of amplification in both of vertebrates and invertebrates,and HT events in vertebrates.Phylogenetic relationship and sequence similarity matrix analysis showed that DD41D/VS was more closely related to DD37D/maT and DD34D/mariner than other families,while DD40D was derived from the DD41D family,rather than an independent evolutionary clade.Insertion age analysis showed that the evolutionary dynamics of VSs in vertebrates were significantly different.Ray-finned fish and toads were young insertions,followed by bats and marsupials,while jawless fishes and lizards mainly repreaented by ancient VS invasions.Cell tests showed that the structurally intact VS in the bat genome has lost transposition activity.2.Based on the annotation of the zebrafish genome,we identified ten autonomous and seventy-seven non-autonomous transposons of Tc1/mariner superfamily,accounting for 5.41%of the zebrafish genome.Phylogenetic analysis showed that the eight autonomous transposons(Mariner-7_DR,Mariner-13-DR,Mariner-15_DR and Tc1-21_DR)belong to the DD34E/Tc1 family,while Mariner-14 DR belongs to the DD37E/TRT family,and Mariner-18 DR belongs to DD×D/pogo family.All these autonomous transposases had conserved DNA binding domains and catalytic domains.Among them,Tcl-8B DR(named as ZB)was 1597 bp in length,contained a protein gene encoding 341 aa transposase and flanked by 201 bp terminal inverted repeats(TIRs).The target site repeat sequence(TSD)of ZB is TA.In the zebrafish genome,there were 25 copies of ZB,and 20 of them encoded intact transposase with identity of more than 99%.Insertion age analysis showed that ZB was the youngest and maybe highly active.3.Cell analysis showed that ZB was more active than other three popularly used transposons(SB,PB,and Tol2)(p<0.05),and ZB had overproduction inhibition(OPI)like others tranposons,but the OPIs of them existed cell and dosage differences.All four transposons showed OPIs in HepG2 cells when the transposon was controlled at low(10 ng)dosage and in Hela cells at a high dosage(500ng).However,the OPIs were not observed for ZB and Tol2 when transfected at high dosge(500ng)in HepG2,while SB and PB exhibited OPIs.ZB left "footprints" of three bases,"CAG" or "CTG",which was in accordance with previous reports in SB and Frog Prince transposon.Integration analysis showed that ZB transposon inserted into the genome non-randomly and has a certain preference for gene regulatory sequences.And prefer to insert a short,palindromic AT-repeat(ATATATAT).4.We designed and constructed enhancer trapping(ET)vectors mediated with the ZB and Tol2 transposon systems in zebrafish,and 60 ZB ET lines and 87 Tol2 ET lines were obtained by microinjection.The efficiency of enhancer trapping mediated with ZB was similar to that of Tol2,which was screened by fluorescence microscope.The germline transfer efficiency of ZB and Tol2 was 55.56%(n=108)and 55.77%(n=156),respectively.ZB tended to produce single GFP expression pattern offsprings than Tol2(p<0.05),while Tol2 tended to produce offsprings with multiple GFP expression patterns(pattern number?2).Four ZB ET lines(ZK32,ZK36,ZK47 and ZK68)and two Tol2 ET lines(TK1 and TK4)were successfully annotated by Sp-PCR.A total of 13 potential enhancers were annotated by VISTA program,and 8 of them were further verified the enhancer activity.5.We designed a mouse enhancer trapping vector and constructed a mice sperm mutant library.By microinjection,ZB transposon and transposase transgenic mice were generated separately,and four double transgenic male mice(sperm mutant library)containing both transposon and transposase transgenes were obtained.Then they corssed with wild FVB mouse to generate the mutants.Through general PCR and Linker-PCR,a total of 135 pups from 12 litters were screened,and 72 ZB transposon-positive mice were obtained.The integration sites of 61 offspring were same to that of the founders,while ZBs in eleven offsprings inserted into new genomic sites.The re-transposition efficiency of ZB was 15.3%(11/72),which was higher than that of SB and PB(about 10%).Three GFP-positive mice(n15,n19,and n111)were obtained,and 11 potential enhancers were obtained by VISTA annotation.In general,the current study systematically revealed the evolution profile of the DD41D/VS family,which belongs to the Tc1/mariner superfamily,and the structure organization,family components and evolution dynamics of Tc1/mariner transposons in the zebrafish genome were well-defined,and we successfully excavated high active ZB transposon,systematically evaluated its transposition activity and characteristics,and constructed a mouse sperm mutant library mediated with ZB system.The application potential of ZB transposon was also evaluated in the study of enhancer trapping in zebrafish and mice,as important animal models.Totally,these data domestrated that ZB can effectively mediate gene transfer,and has great potential in the research of transgenesis,gene therapy and functional genomics.