PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice.
We designed a new type of polyadenylation-signal (PAS) trap vector system in living mice, the piggyBac (PB) (PAS-trapping (EGFP)) gene trapping vector, which takes advantage of the efficient transposition ability of PB and efficient gene trap and insertional mutagenesis of 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 protamine 1 (Prm1) promoter-driven PB transposase transgenes (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 trap insertions 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 mutagenesis in mice.
Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.|Department of vascular surgery, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.|Key Laboratory of Arrhythmias of the Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai, China.
Limei
L
Li
Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.|Department of Cardiology, East Hospital, Tongji University School of Medicine, Shanghai, China.
Peng
P
Liu
Department of Endocrinology, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, PR China.
Liangliang
L
Sun
Department of vascular surgery, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.
Bin Zhou
Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.|Metastasis research institute, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.|School of Life Science and Technology, Tongji University, Shanghai, China.|Shanghai Research Center for Model Organisms, Shanghai, 201203, China.
Jian
J
Fei
D000818:Animals; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D016254:Mutagenesis, Insertional / Q000379:methods*; D026723:Polyadenylation; D011479:Protamines / Q000235:genetics
D004251:DNA Transposable Elements; C513176:Prm1 protein, mouse; D011479:Protamines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins