Uas cd8 gfp

Manufactured by Bloomington Drosophila Stock Center

Sourced in United States

The UAS-CD8::GFP is a genetic construct that expresses the green fluorescent protein (GFP) fused to the CD8 transmembrane protein under the control of the UAS (Upstream Activating Sequence) promoter. This construct can be used to visualize and study the expression patterns of specific genes or cell types in Drosophila melanogaster.

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22 protocols using uas cd8 gfp

Drosophila Rearing and Genetic Stocks

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All flies were reared on standard cornmeal-based fly food. Unless noted otherwise, during developmental and post-eclosion, flies were raised at 25 °C, ~50% humidity and a 12:12 hr light-dark cycle (1400 ± 200 lx white fluorescent light) in humidity and temperature controlled incubators.
The following stocks were used: Canton-S (BL 64349), tub-Gal80^ts (BL 7019), UAS-CD8:GFP (BL 5130), 20XUAS-IVS-mCD8:GFP (BL 32194), UAS-dMBD-R2-RNAi (BL 30481), and UAS-dMBD2/3-RNAi (BL 35347) were obtained from the Bloomington Stock Center (Bloomington, IN). Drosophila virilis (15010–1051.00) and D. yakuba (14021.0261.38) was received from the Drosophila Species Stock Center (San Diego, CA). Cha-Gal80 was a gift from T. Kitamoto and Jay Hirsh generously provided the Tdc2-Gal4 line.

Gupta T., Morgan H.R., Andrews J.C., Brewer E.R, & Certel S.J. (2017). Methyl-CpG binding domain proteins inhibit interspecies courtship and promote aggression in Drosophila. Scientific Reports, 7, 5420.

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Drosophila Tau Transgenic Protocol

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tub-GAL80^TS, w¹¹¹⁸, UAS-CD8GFP, and UAS-CD8-PARP1-Venus were obtained from the Bloomington Stock Center and repo-GAL4 and UAS-tau^WT (0N4R) were kindly provided by Dr. Mel Feany. Double recombinant control (repo-GAL4,tub-GAL80^TS) and triple recombinant tau transgenic (repo-GAL4,tub-GAL80^TS,UAS-tau^WT) flies were balanced over TM6B,Tb. Crosses were performed at 18°C or 28°C in incubators with humidity control and 12-hour light:dark cycles. Flies were maintained on standard Drosophila Bloomington food (Nutri-Fly, Genesee Scientific).

Scarpelli E.M., Trinh V.Y., Tashnim Z., Krans J.L., Keller L.C, & Colodner K.J. (2019). Developmental expression of human tau in Drosophila melanogaster glial cells induces motor deficits and disrupts maintenance of PNS axonal integrity, without affecting synapse formation. PLoS ONE, 14(12), e0226380.

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Drosophila Larval Neuronal Imaging

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Flies were cultured on standard medium at room temperature ∼22°C. Actively crawling third instar larvae of both sexes were used for all the experiments. The GAL4/UAS system was used to drive neuronal expression of all the transgenes (Brand and Perrimon, 1993 (link)). The stock expressing mCherry-tagged Synaptogyrin (SG-mCherry) was kindly provided by Dr. J. T. Littleton (Stevens et al., 2012 (link)). Syn null mutant (cantonized syn⁹⁷) was kindly provided by Dr. Erich Buchner (Godenschwege et al., 2004 (link)). The elav^c155-GAL4, UAS-CD8-GFP, and sei^ts2stocks were obtained from Bloomington Stock Center.

Vasin A., Sabeva N., Torres C., Phan S., Bushong E.A., Ellisman M.H, & Bykhovskaia M. (2019). Two Pathways for the Activity-Dependent Growth and Differentiation of Synaptic Boutons in Drosophila. eNeuro, 6(4), ENEURO.0060-19.2019.

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Drosophila Genetics and Imaging Protocol

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All flies were raised at 25 °C with 12 h/12 h light/dark cycle unless noted. This study: htl-LexA, pyr-Gal4/CyO, htl>FRT>stop>FRT>Gal4, UAS-Pyr:GFP, UAS-Ths:GFP and LexO-Htl:mCherry. All new transgenic injections were performed by Rainbow Transgenic Flies, Inc. Bloomington Drosophila Stock Center: UAS-CD8:GFP, UAS-CD8:RFP, UAS-mCherryCAAX, LexO-CD2:GFP, UAS-Eb1:GFP, UAS-Lifeact:GFP, UAS-nls:GFP, UAS-nls:mCherry, htl-Gal4, ths-Gal4/CyO, UAS-Dia:GFP, UAS-ΔDAD-Dia:GFP, UAS-pyrRNAi, UAS-diaRNAi, hs-Flp, {nos-Cas9}ZH-2A, and w¹¹¹⁸. Vienna Drosophila Resource Center: htl:GFP^fTRG, UAS-htlRNAi, and UAS-thsRNAi. Other sources: LexO-nsyb:GFP^1–10, UAS-CD4:GFP¹¹^{20 (link)}. LexO-mCherryCAAX^{15 (link)}. dpp-Gal4/CyO, LexO-Fz:mCherry and 1151-Gal4 from Huang et al.^{36 (link)} (also see Supplementary Table 3).

Patel A., Wu Y., Han X., Su Y., Maugel T., Shroff H, & Roy S. (2022). Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche. Nature Communications, 13, 1185.

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Drosophila Model of Alzheimer's Disease

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Fly crosses were conducted and maintained at 25 °C, ~60% humidity in diurnally controlled environments. Crosses were conducted in standard cornmeal media. On the day that adults eclosed from the pupal case, they were switched to instant fly media supplemented with vehicle (ultrapure water) or with final 1 mg/mL D-520 dissolved in ultrapure water. Flies were maintained in media for 14 days, with food changed every 3–4 days. On day 14, fly heads were dissected and GFP fluorescence was examined and photographed with an Olympus BX53 microscope. Fluorescence from fly eyes was then quantified and tabulated as described before^{37 (link),44 (link)}. Aβ_1–42 stock was #32038 from Bloomington Stock center. Ctrl flies had the GMR-Gal4 driver and UAS-mCD8-GFP on the w¹¹¹⁸ background. GMR-Gal4 was #8121 from Bloomington stock center and UAS-CD8-GFP was stock #5137 from Bloomington stock center. UAS-mCD8-GFP and GMR-Gal4 were maintained as a trans-heterozygous stock to cross to w¹¹¹⁸ or UAS-Aβ_1–42.

Yedlapudi D., Xu L., Luo D., Marsh G.B., Todi S.V, & Dutta A.K. (2019). Targeting alpha synuclein and amyloid beta by a multifunctional, brain-penetrant dopamine D2/D3 agonist D-520: Potential therapeutic application in Parkinson’s disease with dementia. Scientific Reports, 9, 19648.

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Drosophila Rearing and Genetic Stocks

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Fly stocks were reared on standard cornmeal media (per 1L H₂0: 12g agar, 29g Red Star yeast, 71g cornmeal, 92g molasses, 16mL methyl paraben 10% in EtOH, 10mL propionic acid 50% in H₂0) at 25°C with 60% relative humidity and entrained to a daily 12hr light, 12hr dark schedule. Canton-S flies were from Gero Miesenböck (University of Oxford), w¹¹¹⁸ flies were from David Krantz (UCLA), LexAOP-CD8::GFP; UAS-flp, PBac{y[+mDint2]w[+mc]=brp(FRT.Stop)V5–2A-LexA-VP16} [52 (link)] was from Lawrence Zipursky (UCLA), yw,per⁰¹ [39 (link)] and yw; tim⁰¹ [38 (link)] were supplied by Amita Sehgal (University of Pennsylvania), and UAS-hiw^ΔRING [59 (link)] was from Aaron Diantonio (Washington University in St. Louis). UAS-hiw^RNAi [87 (link)] was ordered from the Vienna Drosophila Resource Center. w⁺, per⁰¹ [39 (link)], w⁺;tim⁰¹ [38 (link)], cyc⁰¹ [37 (link)], Clk^JRK [36 (link)], OR22a-GAL4 [20 (link)], UAS-kir2.1::GFP [44 (link)], tubP-GAL80^TS [46 (link)], UAS-CD8::GFP [88 (link),89 (link)], para^sbl−1 and para^sbl−2 [47 (link)], orco-GAL4 [45 (link)], hiw^ΔN [60 (link)], hiw^ND8 [59 (link)], and UAS-bsk^DN were obtained from the Bloomington Drosophila Stock Center.

Singh P, & Donlea J.M. (2020). Bidirectional regulation of sleep and synapse pruning after neural injury. Current biology : CB, 30(6), 1063-1076.e3.

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Genetic Manipulation of Drosophila

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Flies were kept at room temperature or 25°C. The following fly lines were used w⁻;nrv2-Gal4;nrv2-Gal4, apontic-Gal4, mCherry^dsRNA (BL35785), UAS-CD8-GFP, nanos-Cas9^attP2A (BL36046) (Bloomington Drosophila stock center), PBac{681.P.FSVS-1}MFS3^CPTI002305 (Kyoto stock center), pippin-dsRNA: w¹¹¹⁸; P{GD4548}^v10598 (VDRC), repo-Gal4; repo-Gal4, alrm-Gal4; alrm-Gal4, gli-Gal4 (Christian Klämbt), moody-Gal4 (Stork et al., 2008 (link)), 46F-Gal4 (Hummel et al., 2002 (link)), MFS ^{dsRNA4726R−3} (Japanese National Institute of Genetics), UAS-FLII¹²Pglu-700μδ6 (Volkenhoff et al., 2018 (link)). The dsRNA-constructs used in the RNAi screen are indicated in Supplementary Table 1 and were obtained from Bloomington Drosophila stock center, VDRC or the National Institute of Genetics (NIG).

McMullen E., Weiler A., Becker H.M, & Schirmeier S. (2021). Plasticity of Carbohydrate Transport at the Blood-Brain Barrier. Frontiers in Behavioral Neuroscience, 14, 612430.

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Generating Transgenic Drosophila Lines Expressing Prion Proteins

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Flies expressing RaPrP-WT were described previously (Fernandez-Funez et al., 2010). The constructs carrying RaPrP-S174N, EqPrP-WT, EqPrP-SE167,168DQ, CaPrP-WT, CaPrP-D159N, and CaPrP-YD155,159DN cDNAs were synthesized at GenScript and cloned between EcoRI and NotI sites onto the pUAST Drosophila expression vector (Brand and Perrimon, 1993 (link)). The pUAST-based constructs were injected into yw embryos at Rainbow Transgenics following standard procedures (Rubin and Spradling, 1982 (link)) to generate multiple independent transgenic lines for each plasmid. The driver strains OK107-Gal4 (mushroom bodies), BG380-Gal4 (motor neurons), and da-Gal4 (ubiquitous), and the reporters UAS-LacZ and UAS-CD8-GFP were obtained from the Bloomington Drosophila Stock Center (http://fly.bio.indiana.edu). Fly stocks were maintained on standard Drosophila medium at 25°C. For experiments, homozygous females for the Gal4 strains were crossed with UAS males to generate progeny expressing PrP in the desired tissue. Crosses were placed at 25°C for two days, transferred to 28°C until the progeny completed development, and adults were aged at 28°C, unless otherwise indicated. All assays were performed using females.

Sanchez-Garcia J, & Fernandez-Funez P. (2018). D159 and S167 are protective residues in the prion protein from dog and horse, two prion-resistant animals. Neurobiology of disease, 119, 1-12.

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Fly Genetics: Detailed Protocols

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The following fly stocks were used: Oregon R, w¹¹¹⁸, Ubx¹/TM6B, Tb, Sb, Dfd-lacZ and UAS-Ubx (kindly provided by L. S. Shashidara, IISER, Pune, India), Ubx^6.28/TM6B, Tb, Sb, Dfd-lacZ, abdA^MX1/TM3, Sb, Kr-Gal4, UAS-GFP (Sánchez-Herrero et al., 1985 (link)), Hb9-Gal4/TM3, Sb, ftz-lacZ (Broihier et al., 2002 (link)), 24B-Gal4, UAS-mCherry/TM6B (kindly provided by S. Merabet, IGFL, Lyon, France), Mef2-Gal4, UAS-CD4::td-tom.FP/TM6B (kindly provided by O. Vef, University of Mainz, Germany), Act5C-Gal4/CyO, Wnt4^EMS23, bw/CyO, hb-lacZ, arm⁴/FM7, grh-lacZ, arm⁸/FM7c,Dfd-GMR-nvYFP, UAS-mCherry RNAi, UAS-Ubx RNAi, UAS-arm RNAi, UAS-sgg.B (UAS-GSK3), UAS-dTCFΔN (UAS-dTCF.DN), UAS-CD4::tdGFP and UAS-CD8::GFP (all from Bloomington Stock Center, Indiana, USA). arm⁸/X or arm⁸/X;; Ubx¹/+ animals were identified by the anti-Sex lethal signal.
The UAS-Ubx RNAi insertion on the second chromosome (attP40) was generated using the shUbx RNAi (HMS01403) construct in pValium20 (Ni et al., 2011 (link)) (kindly provided by the TRiP consortium, Harvard, USA). All experiments were performed at 25°C except for the RNAi and dominant-negative experiments, which were incubated at 29°C.

Hessinger C., Technau G.M, & Rogulja-Ortmann A. (2017). The Drosophila Hox gene Ultrabithorax acts in both muscles and motoneurons to orchestrate formation of specific neuromuscular connections. Development (Cambridge, England), 144(1), 139-150.

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Drosophila Genetic Toolkit for Cell Biology

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srpHemo-GAL4 (mac>) was provided by K. Brückner (UCSF, USA) [9 (link)]. Oregon R (control), P{CaryP}attP2 (control), P{CaryP}attP40 (control), kay²(Dfos²), (UAS-Fra)2 (Dfos), UAS-Rho1.N19 Rho1DN), UAS-fbz (DfosDN), UAS-kayak RNAi (Dfos RNAi) TRiP HMS00254 and TRiP JF02804, UAS-dia RNAi TRiP HM05027, UAS-LamC RNAi TRiP JF01406 and TRiP HMS00308, e22c-GAL4 (ecto>), Resille::GFP, UAS-GFP::nls, UAS-dia::EGFP, UAS-diaRBD::EGFP, UAS-mCherry::nls, UAS-CD8::GFP lines were obtained from the Bloomington Stock Center (Indiana, USA). kay¹ (Dfos¹) line was provided by O. Schuldiner (WIS, Israel). UAS-dia::deltaDad::EGFP (diaCA) and srpHemo-GAL4 UAS-CLIP::GFP (mac>CLIP::GFP) lines were provided by B. Stramer (KCL, UK). UAS-cher::FLAG (cher) line was provided by M. Uhlirova (CECAD, Germany). w[1118] (control), UAS-cher RNAi KK107451, UAS-TM4SF RNAi KK102206, UAS-Lam RNAi¹ GD45636, UAS-Lam RNAi² KK107419 lines were obtained from the Vienna Drosophila Resource Center (Austria).

Belyaeva V., Wachner S., Gyoergy A., Emtenani S., Gridchyn I., Akhmanova M., Linder M., Roblek M., Sibilia M, & Siekhaus D. (2022). Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila. PLoS Biology, 20(1), e3001494.

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