Expression-independent gene or polyadenylation[poly（A）]trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed.Although a number of poly（A）-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome,further efforts are needed to avoid the 3＇-terminal insertion bias and the splice donor（SD） read-through,and to improve the mutagenicity.Here,we present a Sleeping Beauty（SB） transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding,gene-breaking and large-scale mutagenesis, respectively.The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element（ARE）,which greatly reduced the SD read-through events,except that the internal ribosomal entry site（IRES） element was introduced in front of the SD signal to overcome the phenomenon of 3＇-bias gene trapping.The breaking cassette consisting of an enhanced splicing acceptor（SA）,a poly（A） signal coupled with a transcriptional terminator（TT） effectively disrupted the transcription of trapped genes.Moreover,the Hsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11,which allows the conditional remobilization of a trap insert from a non-coding region.The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells.In addition,the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus,this poly（A）-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals.
