A gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap
system, the gene-trap system allows the recovery only of fly lines whose genes are
inactivated by a P-element insertion, i.e., mutants. In the gene-trap system, the
reporter gene expression reflects precisely the spatial and temporal expression pattern
of the trapped gene. Flies in which gene trap occurred are identified by a two-step
screening process using two independent markers, mini-w and Gal4, each indicating
the integration of the vector downstream of the promoter of a gene (dual tagging),
mini-w has its own promoter but lacks a polyadenylation signal. Therefore, mini-w
mRNA is transcribed from its own promoter regardless of the vector integration site
in the genome. However, the eyes of flies are not orange or red unless the vector
is incorporated into a gene enabling mini-w to be spliced to a downstream exon of
the host gene and polyadenylated at the 3' end. The promoter-less Gal4 reporter is
expressed as a fusion mRNA only when it is integrated downstream of the promoter of
a host gene. The exons of trapped genes can be readily cloned by vectorette RT-PCR,
followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system
provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of
genes responsible for mutant phenotypes, (iii) precise detection of expression patterns
of trapped genes, and (iv) rapid cloning of trapped genes.
