Flies were reared on standard cornmeal food (130 g of yarn agar, 248 g of Baker’s yeast, 1223 g of cornmeal, and 1.5 liters of sugar beet syrup in 20 liters of distilled water) and kept in a 25°C incubator with light-dark cycle. Fly stocks were obtained from the Bloomington Drosophila Stock Center (BDSC) and the Vienna Drosophila Resource Center. For stainings and SING assays, 2- to 5-day-old female flies were kept on control diet or on filter paper soaked with 10% sucrose solution and 50 or 100 μM rotenone or 20 μM CsA. We obtained the UAS-CaMPARI2 and the TH-Gal4 line from A. Schoofs and the UAS mito-ER tether lines from E. Ziviani. Other fly lines used in this study were w1118 (BDSC, no. 6326), elav-Gal4 (BDSC, no. 8760), tubulin-Gal4 (BDSC, no. 5138), mef-Gal4 (BDSC, no. 27390), handC-Gal4 (37 (link)), actin-Gal4 (BDSC, no. 4414), armadillo-Gal4 (BDSC, no. 1561), UAS-Creld-HA (this study), UAS-mCherry-mitoOMM (BDSC, no. 66533), Drp1-HA (BDSC, no. 42208), UAS Pink1.C (BDSC, no. 51648), UAS catalase-RNAi (BDSC, no. 34020), UAS Duox-RNAi (BDSC, no. 33975), UAS-Sod1 (BDSC, no. 24750), UAS ND-39 (BDSC, no. 15821), UAS GFP-KDEL (BDSC, no. 9899), Pink15 (BDSC, no. 51649), and park25 (52 (link)).
Tubulin gal4
Manufactured by BD
Tubulin-Gal4 is a genetic construct that expresses the Gal4 transcription factor under the control of the tubulin promoter. The tubulin promoter drives ubiquitous expression of the Gal4 protein, allowing it to be used as a tool for targeted gene expression in a wide range of cell types.
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4 protocols using tubulin gal4
1
Drosophila Rearing and Genetic Manipulation
2
Drosophila Model of NGLY1 Deficiency
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Stocks were maintained on standard agar-dextrose-yeast medium at 25°C on a 12-h light/dark cycle. The Drosophila ortholog of human NGLY1 is Pngl, referred to here as dNGLY1. dNGLY1pl flies carry an early stop codon in the dNGLY1 gene, and were previously characterized and generously provided by Perlara, PBC [20 (link)]. The dNGLY1pl allele is homozygous lethal and the stock is maintained with the CyO balancer. The UAS-dNGLY1wt and UAS-dNGLY1C303A constructs were provided by Hamed Jafar-Nejad (Baylor College of Medicine) and described previously [28 (link)]. The dNGLY1ΔGAL4 line was provided by Hugo Bellen (Baylor College of Medicine). The followings stocks were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN): Trh-GAL4 (BDSC # 38389), Tubulin-GAL4 (BDSC # 5138), Dopa Decarboxylase-GAL4 (BDSC # 7009), Tyrosine Hydroxylase-GAL4 (BDSC # 8848), UAS-dNGLY1-RNAi (BDSC # 54853), UAS-sgg-RNAi (BDSC # 31308), and attp2 (RNAi control, BDSC # 36303).
3
Drosophila Gametogenesis and Cell Biology
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tubulin-Gal4 (BDSC#5138), UAS-PLCδ-PH-EGFP (BDSC#39693), tGPH (BDSC#8163), Sqh-GFP.RLC (BDSC#57145), Sqh-mCherry.M (BDSC#59024), p(Ubi-p63E-Feo-mCherry)3 (BDSC#59277), bam-Gal4 [51 (link)], nos-Gal4 (BDSC#4937), chif-Gal4 (BDSC#13134), C587-Gal4 (BDSC#67747), EYFP-Rab4 (BDSC#62542), EYFP-Rab6 (BDSC#62544), EYFP-Rab7 (BDSC#62545), EYFP-Rab11(BDSC#62549), UAS-EGFP (BDSC#5430), tub-Gal80ts (BDSC#7108), UASP-Rab11.S25N (BDSC#23261), UAS-mRFP-Anillin (BDSC#52220), rab7 mutant [69 (link)], Rab35 mutant [71 (link)], genomic CPES_V5tag [27 (link)], cpes mutants [27 (link)], UAS-hSMS1 [27 (link)], dcert1[43 (link)], UAS CDase [44 (link)], UAS-CPES active site mutants, UAS-Aedes CPES, UAS-Bombyx CPES (this study).
4
Drosophila Sirt6 Transgenic Lines
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dSirt6 lines were made by cloning the coding DNA sequence of Drosophila Sirt6 (CG6284) into the following constructs: a pUASt-based pTW vector (Drosophila Genomics Resource Center) for “UAS-Sirt6,” pTGW for “UAS-Sirt6-GFP,” and pUASg.attB for “UAS-Sirt6-3R.” UAS-Sirt6 and UAS-Sirt6-GFP were injected (Best Gene) into w1118 flies to create transgenic lines, with insertions on the second chromosome, while UAS-Sirt6-3R was injected into M{3xP3-RFP.attP}ZH-86Fb flies (Bloomington Drosophila Stock Center [BDSC] #24749) to generate a site-specific insertion on the third chromosome. The following additional stocks were obtained from the BDSC: UAS-dMyc (BDSC #9674), dMyc4 (BDSC #64769), tubulin-GAL4 (BDSC #5138), EP-Sirt6 (BDSC #30115), UAS-Sirt6-RNAi (BDSC #34530), s106 (BDSC #8151), and daughterless-GAL4 (BDSC #55850). The tubulinGeneSwitch (“tubGS”) was a kind gift from S. Pletcher, University of Michigan, Ann Arbor, MI.
For all experiments, flies were aged and maintained on 15% dextrose/15% yeast/2% agar food at 25 °C on a 12-h light/dark cycle at 60% relative humidity. For experiments using GeneSwitch drivers, either RU486 dissolved in EtOH at the indicated concentration (Dataset S1 ) or EtOH alone (for controls) was added to the food at 20 mL/L.
For all experiments, flies were aged and maintained on 15% dextrose/15% yeast/2% agar food at 25 °C on a 12-h light/dark cycle at 60% relative humidity. For experiments using GeneSwitch drivers, either RU486 dissolved in EtOH at the indicated concentration (
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