Repo gal4

Manufactured by Bloomington Drosophila Stock Center

Sourced in United States

The Repo-GAL4 is a genetic tool used in Drosophila research. It drives the expression of target genes in glial cells, which are a type of non-neuronal cells in the Drosophila nervous system. The Repo-GAL4 construct contains the regulatory sequences of the reversed polarity (repo) gene, which is specifically expressed in glial cells, fused to the GAL4 transcriptional activator. This allows for the targeted manipulation and study of glial cell function in Drosophila models.

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38 protocols using repo gal4

Genetic Tools for Drosophila Studies

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armadillo GAL4 (w[*]; P{w[+mW.hs]=GAL4-arm.S}4a P{w[+mW.hs]=GAL4-arm.S}4b; Bloomington Stock Center 1561 that has lost the balancer), eyGAL4, GMRGAL4 (generous gift from Dr. Bob Johnston at Johns Hopkins University), UAS-GFP.nls (P{UAS-GFP.nls}14; Bloomington Stock 4775), UAS-GFP-CAP-D3^{61 (link)}, tubPGAL4 (Bloomington Stock 5138), repoGAL4 (generous gift from Dr. Heather Broihier at Case Western Reserve University), elavGAL4 (Bloomington Stock Center 8765), c253GAL4 (w[1118]; P{w[+mW.hs]=GawB}C253; Bloomington Stock Center 6980), dpnGAL4 (Bloomington Stock Center 47456), optixGAL4 (w[1118]; P{y[+t7.7] w[+mC]=GMR30D11-GAL4}attP2; Bloomington Stock Center 48098), UAS-gypsyCLEVR and UAS-gypsy-CLEVR^ΔPBS^{51 (link)}.

Crawford B.I., Talley M.J., Russman J., Riddle J., Torres S., Williams T, & Longworth M.S. (2024). Condensin-mediated restriction of retrotransposable elements facilitates brain development in Drosophila melanogaster. Nature Communications, 15, 2716.

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DGRP Fly Generation and Maintenance

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DGRP flies were generated and are maintained in our laboratories (Mackay et al. 2012 (link); Huang et al. 2014 (link)). We obtained the OK107-GAL4 line from Dr Tanouye (University of California, Berkeley) and the c739-GAL4, elav-GAL4, repo-GAL4 from the Bloomington Drosophila Stock Center. We also obtained Mi{ET1} lines (CG34113^MB04218, CG34113^MB04817, CG42313^MB08581, Cip4^MB03744, cv-c^MB03489, cv-c^MB03717, Dgk^MB10383, Gefmeso^MB10683, Pkc53E^MB02781, Pkcδ^MB00303, RhoU^MB00991, side^MB07679, trio^MB09917) and their co-isogenic control from the Bloomington Drosophila Stock Center. We obtained UAS-RNAi lines (CdGAPr^KK100409, CG30440^KK101642, CG6424^KK107381, Cip4^KK101912, cv-c^K107255, RhoGAP68F^KK102638, RhoU^KK112816, robo^KK108817, rut^KK109441) and the progenitor control y w¹¹¹⁸;P{attP,y⁺w³} line from the Vienna Drosophila Resource Center (Dietzl et al. 2007 (link)). These lines were crossed to elav, repo, or mushroom body–specific GAL4 driver lines to induce targeted gene silencing. All flies were reared on cornmeal–molasses–agar–yeast medium at 25 °C, 70% humidity, and a 12-h light/dark cycle.

Arya G.H., Magwire M.M., Huang W., Serrano-Negron Y.L., Mackay T.F, & Anholt R.R. (2015). The Genetic Basis for Variation in Olfactory Behavior in Drosophila melanogaster. Chemical Senses, 40(4), 233-243.

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Drosophila Genetic Toolkit Protocols

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Flies were maintained using standard conditions. Fly lines repo-Gal4 (BDSC_7415), longGMR-Gal4 (BDSC_8121), elav-Gal4 (BDSC_8760), Hdc^MB07212 (BDSC_25260), R10D10-Gal4 (BDSC_69558), R32H04-Gal4 (BDSC_49734), R29A12-Gal4 (BDSC_49478), and R19C02-Gal4 (BDSC_49282) were provided by the Bloomington Drosophila Stock Center. The mCD8-GFP and 3XPax3-GFP markers and the carT^43A mutant have been described^{25 (link)}. UAS-WNK-RNAi and UAS-fray-RNAi transgenes and their effectiveness in knocking down the relevant target have been described^{10 (link)}. The UAS-Ncc69-RNAi line (VDRC KK106499) was obtained from the Vienna Drosophila Resource Center (VDRC). The Ncc69^r1, Ncc69^r2 and Gli-Gal4; UAS-Ncc69 flies^{13 (link)} were a gift from Dr. William Leiserson (Yale University, New Haven, CT). The MZ709-Gal4 line was a gift from Dr. Hong-Sheng Li (University of Massachusetts Medical Center, Worcester, MA).

Stenesen D., Moehlman A.T., Schellinger J.N., Rodan A.R, & Krämer H. (2019). The glial sodium-potassium-2-chloride cotransporter is required for synaptic transmission in the Drosophila visual system. Scientific Reports, 9, 2475.

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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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Fly Maintenance and Genetic Manipulation

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Flies were maintained on standard fly food (yeast, dextrose, cornmeal and agar) under a 12 h:12 h light:dark cycle at 25 °C. Flies were transferred to fresh food 2–3 times per week. For drug treatment studies, the LXR-agonists LXR-623 (50 μg/ml, Selleckchem, Cat. No. S8390) and GW3964 (50 μg/ml, Selleckchem, Cat. No. S2630), and the antioxidant N-acetylcysteine amide (AD4; 40 μg/ml, Tocris, Cat. No. 5619) were dissolved in standard fly food. The same food batch with equal volume of the vehicle dimethyl sulfoxide (DMSO; Sigma-Aldrich, Cat. No. D8418) was used for the control. For knockdown experiments, the fly lines used were w¹¹¹⁸ KK control and UAS-Arl6IP1-RNAi KK (line numbers 60100 and 104,790, respectively), obtained from the Vienna Drosophila RNAi Centre, www.vdrc.at [22 (link)] and UAS-Orp8-RNAi and UAS-Vap33-RNAi (line numbers 34743 and 27,312 respectively), obtained from the Bloomington Stock Centre [58 (link)]. Tissue-specific expression was achieved by crossing UAS lines to da-GAL4 (Bloomington Drosophila Stock Center (BDSC) no. 84335); [59 (link)], nSyb-GAL4 [10 (link)], OK6-GAL4 [1 (link)], repo-GAL4 (BDSC no. 7415; [74 (link)], or nrv2-GAL4 (BDSC no. 6799; [80 (link)] as indicated in the text. Other fly stocks used were ebony double balancer stock (BDSC no. 3704), mito::GFP (BDSC no. 42737; [61 (link)], tdTomato-Sec61β (BDSC no. 64746), and Rtnl1::YFP [65 (link)].

Byrne D.J., Garcia-Pardo M.E., Cole N.B., Batnasan B., Heneghan S., Sohail A., Blackstone C, & O’Sullivan N.C. (2022). Liver X receptor-agonist treatment rescues degeneration in a Drosophila model of hereditary spastic paraplegia. Acta Neuropathologica Communications, 10, 40.

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Drosophila Circadian Rhythm Mutants

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The following strains were used in this study: w¹¹¹⁸, Clk^Jrk, Pdf-Gal4, UAS-TrpA1, tubulin-Gal80^ts, Δ263b-Gal4/TM6B, Δ263a-Gal4/CyO, Bereft²⁴ [50 (link)], UAS-Bx and Bx loss-of-function mutation (Bx^hdpR26) [60 (link)]; UAS-mCD8::GFP, Bx-Gal4, Repo-Gal4, UAS-263b, and miR-263b^KO (obtained from the Bloomington Stock Center). All flies were raised on standard cornmeal/agar medium at 25 °C under 12-h:12-h LD cycle.

Nian X., Chen W., Bai W., Zhao Z, & Zhang Y. (2019). miR-263b Controls Circadian Behavior and the Structural Plasticity of Pacemaker Neurons by Regulating the LIM-Only Protein Beadex. Cells, 8(8), 923.

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Drosophila Husbandry and Genetic Lines

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Flies were reared on standard corn meal media (Bloomington Stock Center) and maintained on a 12-hour light/dark cycle at 2°C. repo-GAL4 was obtained from the Bloomington Drosophila Stock Center (BDSC # 7415, RRID:BDSC_7415), elav-GAL4 was provided by Dr. Hugo Bellen (Baylor College of Medicine), and uas-Dube3a was described previously (21 (link)). The GSTD1-LacZ reporter line was provided by Mel Feany and Dirk Bohmann and has been described previously (22 (link)). See Supplemental Table 1 for a complete list of stocks.

Hope K.A., Johnson D., Miller P.W., Lopez-Ferrer D., Kakhniashvili D, & Reiter L.T. (2020). Transcriptomic and proteomic profiling of glial versus neuronal Dube3a overexpression reveals common molecular changes in gliopathic epilepsies. Neurobiology of disease, 141, 104879.

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Genetic Tools for Drosophila Studies

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All fruit fly strains used in this study were cultured using standard fly food in a 25°C incubator unless a different culturing temperature was specifically indicated. The Uba5^KO mutant and the human UAS-UBA5 transgenic fly lines were generated in the Bellen lab (for methods, see below). The Uba5^T2A-Gal4 (#78928), UAS-mCherry.nls (#38424), UAS-FLP (#4540), Uba5^GR (#30359), da-Gal4 (#5460), Act-Gal4 (#4414), elav-Gal4 (#8765), and repo-Gal4 (#7415) lines were obtained from the Bloomington Drosophila Stock Center (BDSC).

Pan X., Alvarez A.N., Ma M., Lu S., Crawford M.W., Briere L.C., Kanca O., Yamamoto S., Sweetser D.A., Wilson J.L., Napier R.J., Pruneda J.N, & Bellen H.J. (2023). Allelic strengths of encephalopathy-associated UBA5 variants correlate between in vivo and in vitro assays. medRxiv.

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Drosophila Strain Collection and Maintenance

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Flies were maintained at 25ºC in 12 h light:12 h dark conditions. D. melanogaster wild-type was w¹¹¹⁸, unless noted otherwise. D. sechellia wild-type was #14021-0248.25 (Drosophila Species Stock Center, UCSD). The sequenced region of Ir75a shown in Fig. 1f was amplified from the following strains (from the Drosophila Species Stock Center, unless noted otherwise): D. sechellia: #14021-0248.08, #14021-0248.11, #14021-0248.13, #14021-0248.15, #14021-0248.19, #14021-0248.25, #14021-0248.27, #14021-0248.30; D. simulans: #14021-0251.195, #14021-0251.196, #14021-0251.197, as well as a Seychelles-isolated D. simulans33 (link). Other published mutant and transgenic lines used were: Ir84a^Gal434 , Ir75a-Gal411 (link), UAS-CD8:GFP35 (link), Mi{ET1}Ir75a[MB00253]36 (link), Df(3L)BSC415 (Ir75a deficiency)37 (link), actin5C-Gal438 (link), elav-Gal4 (Bloomington #458), repo-Gal4 (Bloomington #7415), RAL441, RAL70739 (link) and Tasmania T0940 (link).

Prieto-Godino L.L., Rytz R., Bargeton B., Abuin L., Arguello J.R., Dal Peraro M, & Benton R. (2016). Olfactory receptor pseudo-pseudogenes. Nature, 539(7627), 93-97.

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Genetic Manipulation of Drosophila Frágile X Protein

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Fly stocks were maintained at 25°C on standard cornmeal agar medium. Transheterozygous larvae were produced by crossing w¹¹¹⁸; FRT82B dFmr1³/TM6B GFP (i.e., dFmr1³) with w¹¹¹⁸; FRT82B dFmr1^50M/TM6B GFP (i.e., dFmr1^50M) provided by K. Broadie, Vanderbilt University. Genomic rescue of dFmr1 was performed by crossing dFmr1^50M with w¹¹¹⁸; P[dFmr1]; dFmr1³/TM6C (stock provided by T. Jongens, University of Pennsylvania). For tissue-specific knockdown of dFMR1, w¹¹¹⁸; P[GD1288]v8933, (dFmr1^RNAi-1; from Vienna Drosophila RNAi center) or y¹sc^*v¹; P[y^+t7.7v^+t1.8= TRiP.HMS00248]attP2, (dFmr1^RNAi-2; from Bloomington Drosophila stock center) were crossed with the muscle-specific Gal4 driver w; P[GawB]how^24B (24B Gal4, Bloomington Drosophila stock center), the cardiac-specific Gal4 driver P[tinC-Gal4.Δ4] (TinC Gal4, stock provided by R. Bodmer, Sanford Burnham Medical Research Institute), the neuronal-specific Gal4 driver P{w[+mW.hs] = GawB}elav[C155] (Elav Gal4, Bloomington Drosophila stock center) or the glia-specific Gal4 driver w;repo-Gal4/TM6C (Repo Gal4, Bloomington Drosophila stock center).

Novak S.M., Joardar A., Gregorio C.C, & Zarnescu D.C. (2015). Regulation of Heart Rate in Drosophila via Fragile X Mental Retardation Protein. PLoS ONE, 10(11), e0142836.

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