Drosophila Genetic Manipulation Techniques

Standard Drosophila husbandry procedures were followed. Stocks were maintained on a potato-mash based diet at room temperature. Crosses were performed at 25°C and 60% humidity level. The UAS-ALK-L1198F and UAS-ALK-G1201E constructs were generated using standard cloning techniques and transgenic flies were obtained by injection (BestGene Inc.). white¹¹¹⁸ (w¹¹¹⁸) flies were used as control; GMR-Gal4 (#1104) was received from the Bloomington Drosophila Stock Center at Indiana University (BDSC; NIH P40OD018537). UAS-ALK, UAS-ALK-F1174L, UAS-ALK-I1250T as well as UAS-FAM150A (AUG-β) and UAS-FAM150B (AUG-α) were used for ectopic expression studies in the developing Drosophila eye employing the GAL4-UAS system. Staining of imaginal discs was described previously [38 (link)]. The rabbit monoclonal ALK (D5F3) antibody was purchased from Cell Signalling and employed at a 1:200 dilution. The hybridoma, monoclonal mouse antibody 4F3 (anti-discs large; employed at 1:500), deposited by Corey Goodman was obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the NICHD of the NIH and maintained at The University of Iowa. Samples were analysed under Zeiss Axio Imager.Z2 and AxioZoom.V16 microscopes. Images were acquired with a Zeiss LSM800 confocal microscope or with an Axiocam 503 colour camera employing ZEN blue edition software.