Full-length ap cDNA was synthesized by RT-PCR with adult fly head RNA and 2 primers, 5′-GCGGCCGCCAAAATGGGCGTCTGCACCGAGGAGCGC-3′ and 5′-TCTAGATTAGTCCAAGTTAAGTGGCGGTGTGC-3′. The PCR product was digested with NotI and XbaI, and cloned into a pBluescript (pBS) II SK(+). The ap cDNA was subcloned into the NotI/XbaI-digested pUAST attB vector [65 (link)]. Constructs of UAS-ap were injected into eggs of PBac{y[+]-attP-9A VK00005 (BL24862).
For the generation of the UAS-FRT-stop-FRT-ap RNAi construct (UAS>stop>ap RNAi), an ap RANi fragment was obtained by PCR from the genomic DNA of UAS-ap RNAi (NIG-fly, 8376R-1). Subsequently, it was inserted into the pUAST attB vector using an In-Fusion HD Cloning Kit (Takara Bio, Japan). The primer sequences used for the PCR are as follows: forward, 5′-AACAGATCTGCGGCCGCATAACGCGCAACCTCGAC-3′; reverse, 5′-ACAAAGATCCTCTAGAGGAACAATGCTCCGACTAG-3′. Next, the FRT-stop-FRT cassette (mCD8::GFP vector (Addgene, 24385) and then cloned into the pUAST attB vector containing the ap RNAi fragment by using an In-Fusion HD Cloning Kit. The primer sequences used for the PCR are as follows: forward, 5′-AGGGAATTGGGAATTCGAAGTTCCTATTCCGAAG-3′; reverse, 5′-ATCTGTTAACGAATTCGAAGTTCCTATACTTTCTAG-3′. The UAS>stop>ap RNAi construct was also injected into eggs of PBac{y[+]-attP-9A}VK00005.
For the generation of the UAS-FRT-stop-FRT-ap RNAi construct (UAS>stop>ap RNAi), an ap RANi fragment was obtained by PCR from the genomic DNA of UAS-ap RNAi (NIG-fly, 8376R-1). Subsequently, it was inserted into the pUAST attB vector using an In-Fusion HD Cloning Kit (Takara Bio, Japan). The primer sequences used for the PCR are as follows: forward, 5′-AACAGATCTGCGGCCGCATAACGCGCAACCTCGAC-3′; reverse, 5′-ACAAAGATCCTCTAGAGGAACAATGCTCCGACTAG-3′. Next, the FRT-stop-FRT cassette (