The P{Act5C(FOFO1.0)bratmiRsGAL4miRs} construct is designed to allow screening for cell non-autonomous effects on tumors generated by the knockdown of the brat gene. Expression from the ubiquitous Act5C promoter is blocked by the presence of two STOP cassettes, flanked by FRT and mFRT71 sites respectively. Each STOP cassette is approximately 1.0kb and contains Hsp70Bb and SV40 polyA terminators. An intron flanked by splice donor and acceptor sites is present downstream of the stop cassettes; this contains short hairpin artificial microRNAs (miRs) targeted against both brat and the GAL4 driver. Downstream of the intron is the coding sequence for the EGFP fluorescent protein, tagged at the N-terminal end with the Tag:M(mCd8a) membrane targeting signal. Expression from the Act5C promoter should only occur after excision of both the STOP cassettes, which requires two different recombinases; FLP (targets FRT) and FLPm5 (targets mFRT71). However, some read-through is occasionally seen in cases where both of the STOP cassettes have not been removed.
Short hairpin artificial microRNAs (miRs) that target GAL4. Present in transgenic constructs made using a 'FOFO1.0' based plasmid that contains an Act5C promoter; the construct is designed so that expression of the miRs should only occur once both of the FRT and mFRT71 STOP cassettes located downstream of the Act5C promoter have been excised (requires both the FLP and FLPm5 recombinases, which target FRT and mFRT71 respectively). However, some read-through is occasionally seen in cases where both of the STOP cassettes have not been removed.
Short hairpin artificial microRNAs (miRs) that target brat. Present in transgenic constructs made using a 'FOFO1.0' based plasmid that contains an Act5C promoter; the construct is designed so that expression of the miRs should only occur once both of the FRT and mFRT71 STOP cassettes located downstream of the Act5C promoter have been excised (requires both the FLP and FLPm5 recombinases, which target FRT and mFRT71 respectively). However, some read-through is occasionally seen in cases where both of the STOP cassettes have not been removed.
