PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice.

We designed a new type of polyadenylation-signal (PAS) trapvector system in living mice, the piggyBac (PB) (PAS- trapping(EGFP)) gene trappingvector, which takes advantage of the efficient transposition ability of PB and efficient gene trapand insertional mutagenesisof PAS- trapping. The reporter gene of PB(PAS- trapping(EGFP)) is an EGFP gene with its own promoter, but lacking a poly(A) signal. Transgenic mouse lines carrying PB(PAS- trapping(EGFP)) and protamine1 (Prm1) promoter-driven PB transposasetransgenes (Prm1-PBase) were generated by microinjection. Male mice doubly positive for PB(PAS- trapping(EGFP)) and Prm1-PBase were crossed with WT females, generating offspring with various insertion mutations. We found that 44.8% (26/58) of pups were transposon-positive progenies. New transposon integrations comprised 26.9% (7/26) of the transposon-positive progenies. We found that 100% (5/5) of the EGFP fluorescence-positive mice had new trapinsertions mediated by a PB transposon in transcriptional units. The direction of the EGFP gene in the vector was consistent with the direction of the endogenous gene reading frame. Furthermore, mice that were EGFP-PCR positive, but EGFP fluorescent negative, did not show successful gene trapping. Thus, the novel PB(PAS- trapping(EGFP)) system is an efficient genome-wide gene-trap mutagenesisin mice.