pBacMCS-UAS-SV40 was generated by subcloning a BglII-ApaI fragment containing the SV40 terminator amplified by the polymerase chain reaction using the primers (forward:
Uas dtrpa1
Manufactured by Bloomington Drosophila Stock Center
Sourced in United States
The UAS-dTrpA1 is a genetic tool used in Drosophila (fruit fly) research. It is a transgenic construct that allows for the temperature-dependent activation of the dTrpA1 ion channel, which can be used to manipulate neuronal activity in a spatially and temporally controlled manner.
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Lab products found in correlation
Gad1 gal4, by Bloomington Drosophila Stock Center (2 mentions)
Uas hid, by Bloomington Drosophila Stock Center (2 mentions)
Uas shibirets1, by Bloomington Drosophila Stock Center (2 mentions)
Uas kir2, by Bloomington Drosophila Stock Center (1 mentions)
W1118, by Bloomington Drosophila Stock Center (1 mentions)
Uas mcd8 gfp, by Bloomington Drosophila Stock Center (1 mentions)
Nsyb gal4, by Bloomington Drosophila Stock Center (1 mentions)
Ok107 gal4, by Kyoto Stock Center (1 mentions)
Uas dork1 δnc, by Bloomington Drosophila Stock Center (1 mentions)
Tdc2 gal4, by Bloomington Drosophila Stock Center (1 mentions)
Tβh gal4, by Bloomington Drosophila Stock Center (1 mentions)
Uas chr2, by Bloomington Drosophila Stock Center (1 mentions)
Canton s, by Bloomington Drosophila Stock Center (1 mentions)
Gr66a gal4, by Bloomington Drosophila Stock Center (1 mentions)
13xlexaop2 ivs gcamp6s p10, by Bloomington Drosophila Stock Center (1 mentions)
Uas rpr, by Bloomington Drosophila Stock Center (1 mentions)
Uas campari, by Bloomington Drosophila Stock Center (1 mentions)
9 protocols using uas dtrpa1
1
Generation of Transgenic Silkmoths Using pBacMCS-UAS Vectors
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pBacMCS-UAS-SV40 was generated by subcloning a BglII-ApaI fragment containing the SV40 terminator amplified by the polymerase chain reaction using the primers (forward:5′-GCAGATCTTCAGCCATACCACATTTGTAGA-3′ and reverse: 5′-GCGGGCCCTGAGTTTGGACAAACCACAACT-3′ ) from pBac-UAS-SV40/3xP3EGFP into pBacMCS-UAS[42] (link). For the UAS-mCD8GFP, UAS-GFP.nls, and UAS-dTrpA1 constructs, NotI-NotI polymerase chain reaction fragments containing mCD8GFP, GFP.nls, and dTrpA1 were subcloned immediately downstream from the UAS of pBacMCS-UAS-SV40. The AscI-AscI fragment containing the fibroin L chain (FibL) promoter, EGFP, and FibL 3′UTR amplified by polymerase chain reaction using the primers (forward: 5′-GCGGCGCGCCGGTACGGTTCGTAAAGTTCA-3′ and reverse: 5′-GCGGCGCGCCTATATGGTATTATCGAATAC-3′ ) was subcloned into these constructs to generate pBac[UAS-mCD8GFP-SV40], pBac[UAS-GFPnls-SV40], and pBac[UAS-dTrpA1-SV40], respectively (Figures S1 , S2 ). pUAST-mCD8GFP[37] (link) and D. melanogaster carrying UAS-GFP.nls and UAS-dTrpA1 (Bloomington Drosophila Stock Center, Bloomington, IN) were used as gene sources. Transgenic silkmoths were generated by the piggyBac-mediated germ-line transformation methods, as described previously [27] (link). At least three independent lines were generated and analyzed for each strain.
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pBacMCS-UAS-SV40 was generated by subcloning a BglII-ApaI fragment containing the SV40 terminator amplified by the polymerase chain reaction using the primers (forward:
2
Drosophila Husbandry and Fly Lines
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Flies were raised on standard food containing molasses, cornmeal, and yeast at 25°C under a 12-h:12-h LD cycle. Adult flies, 3- to 5-day-old at the start of experiments, were used. Some experiments included males and females, whereas others included only females, as indicated in figure legends. iso31 (w1118), Canton-S (CS), and per01 (Konopka and Benzer, 1971 (link)) lines were obtained from Amita Sehgal, and CSx-iso31 was generated by replacing the X chromosome of iso31 with the X chromosome of CS. Fly lines carrying nan-Gal4 (#24903) (Kim et al., 2003 (link)), UAS-hid (#65403) (Zhou et al., 1997 (link)), UAS-dTrpA1 (#26263) (Hamada et al., 2008 (link)), and Gad1-Gal4 (#51630) (Hamasaka et al., 2005 (link)) were obtained from the Bloomington Drosophila Stock Center. DATfmn mutants were obtained from Kazuhiko Kume (Kume et al., 2005 (link)), nAChRα4rye from Amita Sehgal (Shi et al., 2014 (link)), and Tbhnm18 from Maria Monastirioti (Monastirioti et al., 1996 (link)). sssP1 (Koh et al., 2008 (link)) and taras132 (Afonso et al., 2015 (link)) were described previously. Transgenic flies carrying UAS-Gad1 shRNA were generated in this study as described below. Fly lines were outcrossed to iso31 for at least five generations, except for CS and CSx-iso31.
3
Investigating Drosophila Circadian Rhythms
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Flies were raised in a 12 h:12 h light:dark (LD) cycle at 25°C in vials containing standard cornmeal medium. For these experiments, we use the following stocks: w1118 (RRID:BDSC_5905), UAS-Kir2.1 (Nitabach et al., 2002 (link)), UAS-dTrpA1 (Rosenzweig et al., 2008 (link)), UAS-mCD8GFP, Clk856-GAL4 (Gummadova et al., 2009 (link)) and OK107-GAL4 (MB) driver) that were obtained from the Bloomington Stock Center. We used the same group of enhancer trap lines used by Gorostiza et al. (2014) (link): 3-86-GAL4, 11-8-GAL4, 4-12-GAL4, 4-93-GAL4, 5-133-GAL4, 4-59-GAL4, 5-43-GAL4 and 7-49-GAL4. These lines were a gift from U. Heberlein (Janelia Farm, USA). For the optical imaging experiments we used the following fly lines: pdf-LexA (Shang et al., 2008 (link)), UAS-GCaMP3 (Tian et al., 2009 (link)), LexAop-P2X2, pdf-GAL4 (Renn et al., 1999 (link)) (RRID:BDSC_6900), Clk4.1-GAL4 (Zhang et al., 2010 (link)), Clk4.5-GAL4, Mai179-GAL4>pdf-GAL80 and tim-GAL4>pdf-GAL80 (Emery et al., 1998 (link)) (RRID:BDSC_7126). We generated the experimental fly lines crossing the different GAL4s to the pdf-LexA,UAS-GCaMP3>LexAop-P2X2 line. The UAS-GCaMP3 was obtained from Janelia Farm and the LexAop-P2X2 was a gift from O. Shafer (University of Michigan) (Yao et al., 2012 (link)). All experimental protocols were performed in accordance with relevant guidelines and ethical regulations of our institution.
4
Drosophila Neurogenetics Experimental Setup
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Drosophila melanogaster flies were reared in plastic vials on standard cornmeal food (12 g agar, 40 g sugar, 40 g yeast, 80 g cornmeal per liter) and transferred to fresh food vials every 2 to 3 days. Flies were generally kept at 25°C, 60% to 65% humidity, and exposed to 12-h light and 12-h darkness with light onset at 8 AM. OK107-Gal4 (106098) was obtained from the Kyoto stock center. nSyb-Gal4 (51635), UAS-shibirets1 (44222), UAS-dTrpA1 (26263), UAS-dOrk1.ΔC2 (6586), UAS-dOrk1.ΔNC (6587), and GMR23E10-Gal4 (49032) were received from the Bloomington stock center. c150-Gal4; MB-Gal80 was obtained from Alex Keene (Florida Atlantic University) [114 (link)]. MB-Gal4GS (FlyBase ID: FBtp0015149), UAS- Aβ42Arctic, UAS-Aβ40, 104Y-Gal4 (FlyBase ID: FBti0072312), and MB247-LexA LexAop-Aβ42Arctic were gifted to us by Mark Wu and Andrew Lin (Johns Hopkins University, University of Sheffield). Elav-Gal4GS was obtained from Frank Hirth (King’s College London). Iso31 [115 (link)] was used as a wild-type strain. The experimental lines were generated by crossing the UAS and Gal4 constructs together. At the same time, the parental lines were crossed with Iso31 to have 1 copy of Gal4 or UAS like in the experimental strain. Moreover, Iso31 was always tested in parallel as a control.
5
Drosophila Stocks in Behavioral Assays
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Drosophila stocks used in this study were obtained from the following sources: the tyramine-β-hydroxylase (TβH) null mutant line8 (link), TβhnM18 (supplied by Henrike Scholz, Cologne, Germany), was described previously8 (link). This line was backcrossed several times with w1118, the control line for all corresponding experiments. Tdc2RO54 as well as flies with the matching genetic background were provided by Jay Hirsh (University of Virginia, USA) and have been described previously40 (link). The following transgenic lines were from the Bloomington Drosophila Stock Center: Tβh-GAL4 (#45904), Tdc2-GAL4 (#9313), UAS-dTrpA1 (#26263) and UAS-ChR2 (#9681). Unless otherwise stated, the flies were raised on standard medium at 25 °C with 50–60% relative humidity under a 12:12 h light/dark cycle as described earlier41 (link). All assays utilised virgin females at a density of 20–25 adults per vial.
6
Drosophila Husbandry and Fly Lines
7
Drosophila Genetics: Diverse Fly Lines
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The laboratory stocks w1118, Canton-S, UAS-dTrpA1, norpA, and Ir76b−/− [BL51309] Drosophila lines were obtained from the Bloomington Stock Center. Poxn∆M22−B5∆XB and PoxnFull1 were provided by J. Alcedo59 (link). Orco2 mutant flies were a generous gift from L. Vosshall60 (link). Gr63a1 mutant flies were a gift from A. Ray. 5-HT2APL00052 mutant and 5-HT2A-GAL4 (3299-GAL4) flies were graciously provided by H. Dierick. Ir8a−/−/Ir25a−/− mutant flies were kindly provided by R. Benton. Three species of Drosophila (D. simulans, D. erecta, and D. virilis) were generously provided by P. Wittkopp. All of these strains were maintained on standard food at 25 °C and 60% relative humidity in a 12:12 h light–dark cycle.
8
Genetic Tools for Neuromodulation in Drosophila
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Wild type (OrgR) crossed to UAS-dTrpA1 (Bloomington #26263) served as control in Figs 1 , 2 , 3 and 5 . Hugin-Gal4 (HugS3-Gal4 (ref. 13 (link)), Bloomington# 58769), GR66a-Gal4 (second Chr., gift from K.Scott31 (link)(formerly described as GR66C1)), GR66a-Gal4 (third Chr., Bloomington# 57670 used in Fig. 7b ), Hugin-lexA (Hug1.2-lexA attp40 (ref. 32 (link))), HugPC-Gal4 (see generation of this Gal4-line below), UAS-eNpHR-YFP (Bloomington# 41753, referred here as UAS-YFP in Supplementary Fig. 3 , since this homozygous line together with HugPC-Gal4 was used as fluorescent marker only), UAS-CaMPARI (Bloomington #58761), UAS-rpr;;UAS-hid (UAS-rpr (Bloomington# 5823) crossed homozygous to UAS-hid33 (link)), UAS-shibireTS34 (link), lexAop-CD4::spGFP11 and UAS-CD4::spGFP1-10 were gifts from K. Scott19 (link), 13x LexAop2-IVS-GCamP6s-p10 (Bloomington #44274).
9
Genetic Tools for Drosophila Neuroscience
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The following lines were used: Or83b-Gal4 (Larsson et al., 2004 (link)); Gr-Gal4 collection including Gr68a-Gal42 (Weiss et al., 2011 (link)); Gr68a-Gal41 (Bray and Amrein, 2003 (link)); ppk23-Gal4, Δppk23, and Δppk29 (Thistle et al., 2012 (link)); Voila1 (Balakireva et al., 1998 (link)); NPF-Gal4 and NPFR1-Gal4 (Wu et al., 2003 (link)); c929-Gal4 (Hewes et al., 2003 (link)); oeno-Gal4 and UAS-hid, stinger (Billeter et al., 2009 (link)); tsh-Gal80 (kind gift of Julie Simpson); UAS-GCaMP5G (Akerboom et al., 2012 (link)); ΔTK1, ΔTK2, and UAS-TK (Asahina et al., 2014 (link)); UAS-spGFP and LexAop-spGFP (Gordon and Scott, 2009 (link)); TK-Gal41 (#51975), TK-Gal42 (#51974), TK-Gal43 (#51973), TK-LexA (#54080), UAS-mCD8:GFP, UAS-stinger, UAS-syt.eGFP, UAS-reaper, UAS-dORKΔC, UAS-DTI, UAS-Shibirets1, UAS-dTrpA1 (Bloomington Stock Center, Indiana, USA); UAS-Gr68a-RNAi (13380, 13381 from VDRC, Vienna, AU) and UAS-TK-RNAi (103662 from VDRC). All other stocks used for screening are described in Table 2 .
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