Drosophila melanogaster flies were reared in plastic vials on standard yeast-based medium under a 12:12 light/dark (8 AM:8 PM) cycle and maintained at 25°C with 50% humidity. Adult, 3–5 day-old female flies were used for all experiments and randomly assigned to experimental groups. Fly lines used for behavioral experiments were R23E10-Gal4 (attp2; Bloomington 49032; Bloomington Drosophila Stock Center, Bloomington, Indiana), UAS-CsChrimson-mVenus (attp18; Bloomington 55134; Provided by Janelia Research Campus, Ashburn, Virginia), and Canton-S (Bloomington 64349). For 2-photon experiments, flies with the genotype 10XUAS-Chrimson88-tdTomato (attp18) / +: LexAop-nlsGCaMP6f (VIE-260b); kindly provided by Barry J. Dickson) / +: Nsyb-LexA (attP2), LexAop-PAGFP (VK00005) / R23E10-Gal4 were used. Optogenetically-manipulated fly lines were maintained on food containing 0.2mM all-trans retinal (ATR; Merck, Darmstadt, Germany) for 24 hours prior to assay to allow for sufficient consumption.
Uas cschrimson mvenus
Manufactured by Bloomington Drosophila Stock Center
The UAS-CsChrimson-mVenus is a genetic construct containing the CsChrimson gene, a red-shifted channelrhodopsin, fused to the mVenus fluorescent protein. This construct is designed to be used as a tool for optogenetic manipulation in Drosophila melanogaster.
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5 protocols using uas cschrimson mvenus
1
Optogenetic Manipulation of Fruit Flies
2
Drosophila Behavioral Manipulation and Neuroimaging
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Drosophila melanogaster flies were reared in vials (groups of 20 flies / vial) on standard yeast-sugar-agar based media (1.0–1.5–0.5 gram ratio) under a 12:12 light/dark (8 AM:8 PM) cycle and maintained at 25°C with 50% humidity. Adult, 3–5 day-old female, flies were used for all experiments and randomly assigned to experimental groups. Fly lines used for behavioral and RNA-sequencing experiments include R23E10-Gal4 (attp2; Bloomington 49032; Bloomington Drosophila Stock Center, Bloomington, Indiana) and UAS-CsChrimson-mVenus (attp18; Bloomington 55134; Provided by Janelia Research Campus, Ashburn, Virginia)[31 (link)]. For all 2-photon experiments, flies with the genotype 10XUAS-Chrimson88-tdTomato (attp18) / +: LexAop-nlsGCaMP6f (VIE-260b; kindly provided by Barry J. Dickson) / +: Nsyb-LexA (attP2) [32 (link)], LexAop-PAGFP (VK00005) / R23E10-Gal4 were used. Optogenetically-manipulated fly lines were maintained on food containing 0.5mg/ml all-trans retinal (ATR; Merck, Darmstadt, Germany) for 24 hours prior to assay to allow for sufficient consumption. Pharmacologically-manipulated flies were maintained on food with 0.1 mg/ml of Gaboxadol (4,5,6,7-tetrahyrdoisoxazolopyridin-3-ol, THIP) for the duration of behavioral experiment [23 (link)].
3
Genetic Toolkits for Drosophila Neuroscience
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Strains previously described by Diao et al. (4 (link)) include tubP-Gal4DBD, VGlutMI04979-Gal4DBD, VGlutMI04979–p65AD, and ChaTMI04508–Gal4DBD. UAS-6XmCherry, Gad1MI09277–Gal4DBD, UAS-Cs.Chrimson.mVenus, and UAS-nucLacZ were from the Bloomington Drosophila Stock Center. Repo-Gal80 and dUAS-dTrpA1 were gifts from Tzumin Lee and Paul Garrity, respectively. Transgenic flies created in this study are described in SI Appendix .
4
Drosophila Neuron Manipulation Protocols
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In the present study we used both male and female flies. Flies were raised at 25°C with a 12/12 h light/dark cycle with a humidity level of ∼50%. Wild-type and transgenic flies were obtained from the Bloomington Drosophila Stock Center, Drosophila Genomics Resources Center, and Brain Research Center (BRC) in National Tsing Hua University (NTHU). We used wild-type w+ (BRC, NTHU), ;;UAS-Kir2.1 (Guo et al., 2014 (link); Shuai et al., 2015 (link); BRC, NTHU), ;;VT5404-GAL4 (Lin et al., 2013 (link); BRC, NTHU), ;;UAS-CsChrimson.mVenus (Bloomington stock #55136), ;;UAS-GFP (Bloomington stock #1522), tub-GAL80ts;; (Bloomington stock #7017), c105-GAL4;; (Humberg et al., 2018 (link); Bloomington stock #30822), ;;UAS-TNT (Eisel et al., 1993 (link); Sweeney et al., 1995 (link); Bloomington stock #28997), and ;;ninaE (Drosophila Genomics Resources Center #109599). For the neuron suppression experiments, all GAL4 lines were crossed to the UAS-Kir2.1 or UAS-TNT effector lines and the expression can be controlled by temperature with the combination of tub-GAL80ts. For the optogenetic experiments, all GAL4 lines were crossed to the UAS-CsChrimson.mVenus effector lines. Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author.
5
Drosophila Rearing and Manipulation Protocols
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Drosophila melanogaster flies were reared in vials (groups of 20 flies / vial) on standard yeast-sugar-agar based media (1.0–1.5–0.5 gram ratio) under a 12:12 light/dark (8 AM:8 PM) cycle and maintained at 25°C with 50% humidity. Adult, 3–5 day-old female, flies were used for all experiments and randomly assigned to experimental groups. Fly lines used for behavioral and RNA-sequencing experiments include R23E10-Gal4 (attp2; Bloomington 49032; Bloomington Drosophila Stock Center, Bloomington, Indiana) and UAS-CsChrimson-mVenus (attp18; Bloomington 55134; Provided by Janelia Research Campus, Ashburn, Virginia)[31 (link)]. For all 2-photon experiments, flies with the genotype 10XUAS-Chrimson88-tdTomato (attp18) / +: LexAop-nlsGCaMP6f (VIE-260b; kindly provided by Barry J. Dickson) / +: Nsyb-LexA (attP2) [82 (link)], LexAop-PAGFP (VK00005) / R23E10-Gal4 were used. Optogenetically-manipulated fly lines were maintained on food containing 0.5mg/ml all-trans retinal (ATR; Merck, Darmstadt, Germany) for 24 hours prior to assay to allow for sufficient consumption. Pharmacologically-manipulated flies were maintained on food with 0.1 mg/ml of Gaboxadol (4,5,6,7-tetrahyrdoisoxazolopyridin-3-ol, THIP) for the duration of behavioral experiment [23 (link)].
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