A set of stocks that each contain a Trojan-GAL4 gene trap sequence in a small coding intron, inserted via the CRISPR/Cas-9 drop-in technique described in Kanca et al., 2019, eLife 8: e51539 (FBrf0244062), together with a deletion of coding exons of the targeted gene that are downstream of the inserted gene trap cassette. The inserted cassette acts contains a 'Trojan GAL4' gene trap element composed of a splice acceptor site followed by the T2A peptide, the GAL4 coding sequence and an SV40 polyadenylation signal. When inserted in a coding intron in the correct orientation, the cassette should result in expression of GAL4 under the control of the regulatory sequences of the trapped gene. Deletion of the downstream exons of the targeted gene prevents any mRNA splicing 'over' the Trojan exon that might otherwise occur due to the small size of the targeted coding intron. The strategy uses two gRNAs: one targeted to the coding intron and the other to a non-coding exon in the 3' UTR of the gene. Each line contains one of three versions of the cassette (TI{CRIMIC.TG4.0}, TI{CRIMIC.TG4.1} or TI{CRIMIC.TG4.2}), depending on the frame required to generate a gene trap. In addition, the presence of inverted attP sites flanking the inserted DNA allows for the entire cassette to be replaced with DNA from a compatible donor plasmid (where the sequence to be inserted is flanked by inverted attB sites) through recombination-mediated cassette exchange (RMCE) driven by the phiC31:int integrase.
The PCR check of the insertion gave multiple bands for the left flank and the expected sized product for the right flank. The insertion junction of the right flank was verified by sequencing. The deletion associated with the insertion is 286 bp upstream of the annotated 5' end of the tyf gene and may affect its expression. The other end of the deletion is only 19 bp from the annotated 3' end of the dgt4 gene and may affect proper mRNA 3' end formation.
