Preliminary Study On Enhancer Trapping Mediated By Transposons In Zebrafish

Transposon(Tn),a variety of biological genomes present in the biological world,is a DNA sequence that can move freely on the genome and change its position.Using the naturally occurring gene transfer properties of transposons,many DNA transposons are used as genetic vectors for genomic genetic modification.Sleeping Beauty(SB),PiggyBac(PB)and Tol2 are widely used in vertebrate transgenic manipulation,and the gene mutant library is constructed to study the mechanism and expression of gene expression.In this study,we used the self-constructed SB,PB and Tol2-mediated enhancer trapping vectors to prepare the transgenic zebrafish and compared the reporter gene with the endogenous gene of spatio temporal expression patterns in order to obtain specific enhancers that regulate tissue development or cell differentiation.The main results are as follows:(1)SB,PB,and Tol2-mediated enhancer trapping transgenic zebrafish F1 were classified into seven individuals with typical expression patterns:SK-1,SK-3,SK-6,SK-12,TK-1,TK-4 and PK-0 according to the fluorescent phenotype.The F2 lines were generated by mating the seven lines respectively with the wild type and the expression parttem of green fluorescent protein was observed at different stages in the early stage of embryonic development.The results showed that the phenotypes of the seven lines were mainly divided into brain,trunk,eyes,heart and mandibular.The F2 genome SP-PCR and sequencing results showed that the enhancer trapping vector was integrated into 5 different chromosomes at different loci of the genome.Seven different endogenous genes(slc9a8?ednraa?nettl22?wdr33?dlx1a?rps26 and itgav)were trapped,four of the insertion sites were located in the intron,one in the exon,two at the 20kb upstream of the gene and 10bp downstream.(2)The results showed that the expression pattern of endogenous gene was consistent with the fluorescence signal at two developmental stages of 24hpf and 48hpf,using the antisense RNA of endogenous gene as the probe.Brain,eye and body fluorescence signals such as strong organization after its hybrid color is also deep.In conclusion,this study shows that the three transposon-mediated enhancer trapping vectors are effective for enhancer trapping.The repoter gene and the endogenous gene may be expressed under the same enhancer,so the green fluorescence signal may represent the temporal and spatial expression.This study may provide an important reference for enhancer annotation,which lays the foundation for further study of enhancer expression and gene activity,which is of great significance in the post-genome era.