Appl gal4

Manufactured by Bloomington Drosophila Stock Center

Sourced in Spain

Appl-GAL4 is a genetic tool used in Drosophila research. It is a transgenic fly line that expresses the GAL4 transcriptional activator under the control of the Appl (Amyloid Precursor Protein-like) gene promoter. The Appl-GAL4 driver can be used to direct the expression of target genes or reporter constructs in a specific subset of neurons in the Drosophila brain.

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6 protocols using appl gal4

Genetic Manipulation of Antioxidant Capacity in Drosophila

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Flies were separated by sex and maintained in different vials on a standard sugar-yeast medium at constant temperature (25°C) and humidity (60%) in a 12:12 h light-dark cycle in Binder KBF720-ICH (Binder, Germany) climate chamber. The following strains were used: UAS-Gclc (provided by Dr. William C. Orr, Southern Methodist University) and Appl-GAL4 (#32040, Bloomington Drosophila Stock Center). They were backcrossed with w¹¹¹⁸ (#3605, Bloomington Drosophila Stock Center, USA) 6–8 times to equilibrate the genetic background. To activate the Gclc constitutive overexpression in the nervous system, the UAS-Gclc was crossed with Appl-GAL4 driver. Newly enclosed progenies were maintained for one, four, and 6 weeks before dissection. The same aged flies from the parental UAS-Gclc line were used as a contro control.

Moskalev A., Guvatova Z., Shaposhnikov M., Lashmanova E., Proshkina E., Koval L., Zhavoronkov A., Krasnov G, & Kudryavtseva A. (2019). The Neuronal Overexpression of Gclc in Drosophila melanogaster Induces Life Extension With Longevity-Associated Transcriptomic Changes in the Thorax. Frontiers in Genetics, 10, 149.

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Drosophila Nerve Injury and Transgene Expression

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Flies were grown on standard cornmeal, molasses food at 25°C. Males were typically used. Strains included appl-GAL4, Hemese-GAL4, repo-GAL4, UAS-GAL4, UAS-mCD8-GFP, UAS-mCD8-ChRFP, and LexAop-mCD8-GFP, UAS-bskDN, UAS-hepCA, and UAS-Lifeact-Ruby27 (link)28 (link) (Bloomington Drosophila Stock Center (BDSC), Indiana University (Bloomington, IN)). Other lines were UAS-EB1-RFP (Dr. M. Rolls, U. Michigan, Ann Arbor, MI), LexA-CD4-GFP and LexA/nSynaptobrevin46 (link), elav-GS32 (link), tubulin-GS (Dr. Scott Pletcher, U Michigan, Ann Arbor, MI). Animals were collected over a 2h period then aged 5h for 6h, 24h for 1d, and 72h for 3d animals. Flies on RU486 (Mifepristone, Sigma Aldrich, cat# M8046) were maintained on food supplemented with drug32 (link). Briefly, for each food vial, 50 ul of RU486 stock solution [4.0 mg/ml in 100% ethanol] was pipetted onto the medium and allowed to penetrate the food overnight. UAS-transgenes showed robust expression throughout the wing within 18h of being placed on RU486-containing food, and visible transgene expression (hinge region close to the body of animal) by 6h32 (link)47 (link). Bottles of animals were cleared, collected over a 2h period and put on RU486 or vehicle food and aged for nerve injury.

Soares L., Parisi M, & Bonini N.M. (2014). Axon Injury and Regeneration in the Adult Drosophila. Scientific Reports, 4, 6199.

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Drosophila Genetic Tools for Autophagy

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The following stocks were obtained from Bloomington Drosophila Stock Center: ApplGal4 (#32040), Ddc-Gal4 (#7009), UAS-myrGFP (#32199), UAS-HTT.16Q/CyO (#33810), UAS-HTT.128Q (#33808), w¹¹¹⁸ (#5905), UAS-Hsap\SNCA.A53T (#8148), Mi{MIC}EDTP^MI08496(#44782), Df(2 R)BSC161 (#9596), P{EPgy2}EDTP^EY22967(#22600), cgGal4 (#7011), UAS-GFP-2xFYVE (#42712). UAS-Parkin-R275W/TM3 was kindly provided by Ng Chee Hoe (NNI, Singapore)41 (link), UAS-mCherry-Atg8a48 (link), hsFlp; pAct < CD2 < Gal4,UAS-nlsGFP, r4-mCherry-Atg8a40 (link); hsFlp; r4-mCherry-Atg18a; pAct < CD2 < Gal4,UAS-nlsGFP, and outcrossed Syx17^LL06330 mutants40 (link) were gifts from Gabor Juhasz (Eötvös University, Budapest, Hungary). Fly stocks were raised on standard cornmeal-sugar agar medium at 18–25 °C. L3 feeding larvae were treated for 3 hours prior to dissections. Animals were placed into a suspension consisting of instant yeast medium, supplemented by AUTEN-99 solved in DMSO (Sigma, D8418) or the same volume of DMSO only for untreated samples. For analyzing adult animals, flies were placed into vials containing treated medium immediately after eclosion and kept at 29 °C during the entire experiment. AUTEN-99 dissolved in DMSO was added to yeast suspension (final concentration was 100 or 200 μM), and dropped 65 μl to the surface of each vials. Flies were transferred into a fresh vial in every second day.

Kovács T., Billes V., Komlós M., Hotzi B., Manzéger A., Tarnóci A., Papp D., Szikszai F., Szinyákovics J., Rácz Á., Noszál B., Veszelka S., Walter F.R., Deli M.A., Hackler L J.r., Alfoldi R., Huzian O., Puskas L.G., Liliom H., Tárnok K., Schlett K., Borsy A., Welker E., Kovács A.L., Pádár Z., Erdős A., Legradi A., Bjelik A., Gulya K., Gulyás B, & Vellai T. (2017). The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms. Scientific Reports, 7, 42014.

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Drosophila Genetics and Husbandry Protocol

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The following fly stocks were obtained from Bloomington Drosophila Stock Center: w¹¹¹⁸ (#3605), CS (#64349), UAS-mito-GFP (#8442), Elav-GS-Gal4 (#43642), Daughterless (DA)-GS-Gal4, Appl-Gal4 (#34040). Tsinghua Fly Center: Vimar RNAi (TH1142), Miro RNAi (TH2781), TRip background control (attp2, TB00072). We generated the following lines: hs-GluR1^Lc, UAS-Vimar/tb, and UAS-Vimar/cyo. For all experiments, flies were kept at 25 °C with a 12:12 h light/ dark cycle and constant humidity (65%) on the standard sugar−yeast−agar medium (15 g/l agar, 50 g/l sugar, 100 g/l autolyzed yeast, 6 g/l nipagin and 3 ml/l propionic acid). Flies were raised at standard density in 100 ml bottles unless otherwise stated. All experiments of survival assay and behavior test were carried out with male and female files (10:10 in each tube). Female flies were used in all confocal microscopy imaging experiments.

Xiong Y., Cheng Q., Li Y., Han Y., Sun X, & Liu L. (2023). Vimar/RAP1GDS1 promotes acceleration of brain aging after flies and mice reach middle age. Communications Biology, 6, 420.

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Drosophila Neurodegeneration Genetics

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All fly stocks were maintained and raised under standard conditions at 25°C. Canton S was used as wild type. UAS-SWS^R133A and UAS-PKA-C3 are described in [24] (link), and sws¹ in [19] (link). elav-GAL4, UAS-dicer, and UAS-lacZ were obtained from the Bloomington stock center and Appl-GAL4 was kindly provided by L. Torroja (Universidad Autonoma de Madrid, Spain). The PKA-C3 RNAi line (Pka-C3^NIG.6117R) was obtained from the National Institute of Genetics (NIG-Fly), Japan.

Wentzell J.S., Cassar M, & Kretzschmar D. (2014). Organophosphate-Induced Changes in the PKA Regulatory Function of Swiss Cheese/NTE Lead to Behavioral Deficits and Neurodegeneration. PLoS ONE, 9(2), e87526.

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Drosophila Husbandry and Genetic Manipulation

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Fly husbandry and aging were performed as described (Gargano et al., 2005 (link)). da-Gal4, mef2-Gal4, 188Y-Gal4, appl-Gal4, cisd2^G6528 and all Ppt1 modifiers listed in supplementary material Table S5 were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN). UAS-RNAi transgenic lines for cisd2 (v33925 and v33926), cln3 (v5322) and cln7 (v5089 and v5090) were purchased from the Vienna Drosophila RNAi Center (Vienna, Austria) (Dietzl et al., 2007 (link)). The cisd2 RNAi transgenes do not have predicted off-target effects (defined as genes with at least one continuous stretch of 19 nucleotides complementary to any region of the RNAi transgene (http://stockcenter.vdrc.at/control/vdrcdefinition)). The gmr-Gal4-v33925 and the gmr-Gal4-v33926 double transgenic flies were created by recombining the gmr-Gal4 element and the cisd2 RNAi transgenes onto the same chromosome. UAS-Ppt1 transgenic strains are previously described (Korey and MacDonald, 2003 (link)). Ppt1 loss of function mutant strains (Ppt1^A179T and Ppt1^S77F) were provided by Robert Glaser (Wadsworth Center, Albany NY). The cln3 overexpression strain (UAS-cln3 no. 4) was provided by Richard Tuxworth (Kings College London, London, UK). Sources for all other strains are indicated in supplementary material Tables S3, S4, S5.

Jones M.A., Amr S., Ferebee A., Huynh P., Rosenfeld J.A., Miles M.F., Davies A.G., Korey C.A., Warrick J.M., Shiang R., Elsea S.H., Girirajan S, & Grotewiel M. (2014). Genetic studies in Drosophila and humans support a model for the concerted function of CISD2, PPT1 and CLN3 in disease. Biology Open, 3(5), 342-352.

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