All Drosophila melanogaster stocks, and crosses were maintained using standard medium at 25°C. The fly strains were, srp3 (BL2485), srpAS (BL59020), Tub-Gal4 (BL5138), w1118 (used as wild type background, BL3605), attP2 (BL25710) from the Bloomington Drosophila Stock Center, UAS-dsUsh (GD5712) (from Vienna Drosophila Resource Center) and srp6G (Reuter, 1994 (link)). The strains ushVX22 and ushRev24 were supplied by P. Heitzler. The fly lines msn-mCherry and Tj-Gal4 were kindly provided by the R. Schulz lab and Luisa Di Stefano, respectively.
Attp2
Manufactured by Bloomington Drosophila Stock Center
The AttP2 is a genomic landing site for Drosophila transgenesis. It provides a defined chromosomal location for the integration of transgenes via phiC31 integrase-mediated site-specific recombination.
Automatically generated - may contain errors
6 protocols using attp2
1
Drosophila Genetic Toolkit for Developmental Studies
2
Drosophila RNAi Experiments for Top2 and mei-W68
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Flies were maintained on standard food at 25°C. Fly stocks used for RNAi experiments were y w; spapol, w; {matα4-GAL-VP16}V37 (Bloomington 7063), y1 sc1 v1; P{y[+t7.7] v[+t1.8] = TRiP. GL00338}attP2 (Bloomington 35416) an RNAi construct targeting the Top2 (CG10223) gene, and y v; CG33296 RNAi (described below). To obtain control flies containing only one copy of the driver or RNAi construct, the designated stocks were crossed to y w; spapol flies. Transheterozygotes mutant for mei-W68 (CG7753) were made from the following stocks: y/BS Y; mei-W68Z1049 cn bw/SM6a and y/y+ Y; mei-W68Z4572 cn bw/Cyo[49] (link). The genotype of the nanos-Gal4:VP16 driver flies was y w/y+ Y; nanos-Gal4:VP16; spapol.
3
Drosophila Genetic Manipulation for Heat Stress Study
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Flies were raised on standard cornmeal-agar medium at 25°C in a light/dark cycle of 12 h/12 h. The Hsf 4 mutant was obtained from the Drosophila Genetic Resource Center (stock no. 108–256) and the Hsf 4; Hsf+ rescue line from the Bloomington Stock Center (stock no. 5490, described in ref. 26 (link)). CnBw, which is the genetic background for those lines, was used as the wild-type control in all experiments. The following fly lines have been described previously: Hml-Gal4 (ref. 27 (link)), C564-Gal4 (ref. 28 (link)), UAS-Hsp70 (ref. 29 (link)), UAS-Hsf (ref. 16 (link)). The HsfRNAi line expressing a short hairpin targeting Hsf, and the driver Act-Gal4 were obtained from the Bloomington Stock Center (stock no. 27070 and 4414, respectively). The genetic background of wild-type flies used for RNA sequencing is y1w1, and has been described previously as EHMT+ (ref. 30 (link)) or G9a+/+ (ref. 31 (link)). In vivo RNAi experiments were performed by crossing GMR-Gal4, UAS-thRNAi/CyO virgins32 (link) with HsfRNAi male flies or with control flies containing the attP landing site used to introduce the RNAi-inducing transgene (y1v1; attP2; Bloomington stock no. 36303). The eye phenotype was assessed in three to five-day-old female F1 offspring lacking the CyO balancer.
4
Genetically Engineered Drosophila Constructs
5
Drosophila Transgene Insertion Protocols
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Germline transformation of transgenes was done by BestGene Inc. either by random P-element transformation or site-specific phiC31 methodology (Groth et al., 2004 (link)). The vectors used were w+-marked allowing w+-based selection of recombinants. Specific integration sites attP40 (cyto site 25C6; Markstein et al., 2008 (link)) and attP2 (stock 8622; Bloomington Drosophila Stock Center; Groth et al., 2004 (link)) were used for transgene insertion on second and third chromosomes, respectively. Brp bait variants (CD8::EGFP, brpC-tip and brpC-long) were integrated into attP2 to ensure similar expression conditions.
6
Drosophila melanogaster Rearing and Maintenance
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The experimental model in this study is Drosophila melanogaster. All transgenic lines generated in this study were based on lines attP2 (Bloomington Stock Number: 25710) and VK33 (Bloomington Stock Number: 32543). When outcrosses were needed, we used line w1118 (Bloomington Stock Number: 5905). Information on developmental stages used is described in the main text, figures and/or method details (see below). All fly lines were kept in mite-free conditions, grown at room temperature in the lab or in a room where temperature is set at 25°C, in plastic vials or plastic bottles supplemented with fly food (1.8% m/v yeast, 1% m/v soy flour, 8% m/v cornmeal, 8% m/v malt extract, 1.2% m/v agar, 2.2% m/v sugar beet syrup, 0.625% v/v propionic acid, 1.2% v/v nipagin 20% in ethanol). All fly lines were flipped to new vials/bottles at least every two weeks.
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