The P{Act5C(FOFO2.0)pros^miRsGAL4^miRs} construct is designed to allow screening for cell non-autonomous effects on tumors generated by the knockdown of the pros gene. Expression from the ubiquitous Act5C promoter is blocked by the presence of two STOP cassettes, flanked by FRT and mFRT71 sites respectively. The first STOP cassette is approximately 2.8kb and contains the Lam coding sequence followed by Hsp70Aa and Hsp27 polyA signals. The second STOP cassette is approximately 5.7kb and consists of four tandem SV40 polyA sequences. 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 pros and the GAL4 driver. Downstream of the intron is the coding sequence for the EGFP fluorescent protein, tagged at the C-terminal end with the Tag:NLS(SV40-largeT) nuclear localization 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). After excision of the two cassettes, both the miRs and the fluorescent marker (represented by Avic\GFP^{EGFP.FOFO2.0.Act5C}) are expressed, in the overlap of expression between the FLP and FLPm5 recombinases; typically FLP is expressed using a specific enhancer (to give spatial control) and FLPm5 is expressed using heat shock (to give temporal control). The miRs that target pros (represented by pros^{miR.FOFO2.0.Act5C}) knock down this gene in the the overlap of expression between the FLP and FLPm5 recombinases, inducing a tumor. A second gene of interest can be expressed using the UAS-GAL4 system, to study its effect on tumor formation; the presence of the miRs that target GAL4 (represented by Scer\GAL4^{miR.FOFO2.0.Act5C}) ensures that only cell non-autonomous effects are studied; if the expression pattern of the GAL4 driver used to express the second gene of interest overlaps with that used to produce the tumor, these miRs will knockdown GAL4 expression in the tumor.

Short hairpin artificial microRNAs (miRs) that target GAL4. Present in transgenic constructs made using a 'FOFO2.0' 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).

Short hairpin artificial microRNAs (miRs) that target pros. Present in transgenic constructs made using a 'FOFO2.0' 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).