A new piggyBac-based gene-trap vector, pB-GT1, was constructed. pB-GT1 contains three marker genes, dsRed, Gal4, and EGFP. dsRed is under the control of the constitutive 3xP3 promoter, which induces dsRed expression wherever the vector is inserted in the host genome. The Gal4 sequence has no promoter but is preceded by the splice acceptor site so that it can be transcribed as a transcript fused with the host exon 5′ to the insertion site. EGFP is driven by the constitutive ie+ hr promoter but lacks a poly(A)+ signal sequence, and thus the EGFP expression is detectable only when its transcript is fused with the host exon 3′ downstream of the insertion. By the microinjection of the vector into fertilized eggs, we obtained transgenic Drosophila with a single copy of pB-GT1, which was inserted into the first intron of the ovo gene. The female flies of this transgenic line are sterile, indicating that the insertion inactivated the ovo gene, generating a new allele of this locus, ovopB-GT1. RT-PCR analysis demonstrated that an ovo-Gal4-fusion transcript is produced in ovopB-GT1 flies. The fact that UAS-EGFP reporter expression was detected in ovopB-GT1 germ cells in a pattern similar to that reported for wild-type ovo indicates that functional Gal4 is expressed via pB-GT1, recapitulating the endogenous expression pattern of the trapped gene. pB-GT1 is thus useful in insect genomics for the efficient assignment of functions of individual genes.
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