Ey gal4

Manufactured by Bloomington Drosophila Stock Center

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Ey-Gal4 is a genetic tool used in Drosophila research. It is a transcriptional driver that expresses the yeast Gal4 protein under the control of the eyeless (ey) promoter, allowing targeted gene expression in the developing Drosophila eye.

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7 protocols using ey gal4

Drosophila Strain Maintenance and RNAi

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For most experiments Drosophila strains were cultured in standard cornmeal-agar medium (#789211, NutriFly BF, Genesee Scientific) and maintained at 25 °C. The RNAi experiments were performed at 28 °C as previously described [35 (link)]. The sau^z2217 mutant strain was described previously [35 (link), 67 (link)]. UAS-dGOLPH3 RNAi (#46150, [35 (link)]) and UAS-Tctp RNAi (# 26632) were obtained from the Vienna Drosophila Resource Center Collection (VDRC, [44 (link)]). The following fly strains were from the Bloomington Drosophila Stock Center (BDSC, Indiana University, Bloomington, IN, USA): UAS-Rheb (#9689), UAS-GFP (#4775), Df(2 L)Exel7010 (#7782), en-Gal4 (#30564), nub-Gal4 (#86108), ey-Gal4 (#5534), elav-Gal4 (#8765), and bam-Gal4 (#80579, [68 (link)]). The following fly strains were obtained from FlyORF (University of Zurich, [69 (link)]): UAS-dGOLPH3-HA (#F002769), UAS-Tctp-HA (#F002479), UAS-14-3-3ζ-HA (#F001064). bam-Gal4 and tub-Gal4/tub-Gal80^ts (gift of Dr. Timothy Megraw, Florida State University, USA) were used to deplete dGOLPH3 respectively in spermatocytes and in larval/pupal tissues and to express dGOLPH3-HA, Tctp-HA, and 14-3-3ζ-HA proteins. The fly strains expressing either GFP, or fluorescent tagged-dGOLPH3 were described previously: GFP [34 ], dGOLPH3-RFP [34 ], GFP-dGOLPH3 [35 (link)], and GFP-dGOLPH3^K167A/R170L [35 (link)].

Frappaolo A., Karimpour-Ghahnavieh A., Cesare G., Sechi S., Fraschini R., Vaccari T, & Giansanti M.G. (2022). GOLPH3 protein controls organ growth by interacting with TOR signaling proteins in Drosophila. Cell Death & Disease, 13(11), 1003.

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Drosophila Genetic Stock Maintenance

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Drosophila stocks were maintained on standard cornmeal-molasses food at 25°C. The PINK1-myc transgenic line, UAS-PINK1 transgenic line, UAS-Miro-myc transgenic line, Mhc-GAL4 driver line and Dmef2-GAL4 driver line have been previously described [20] (link), [34] (link), [44] (link), [45] (link), [46] (link). The following lines were obtained from the Bloomington Drosophila Stock Center: elav-GAL4, ey-GAL4, sqh-mito-EYFP, Lon^G3998, Df(3L)Exel9011, P{VALIUM20-mCherry}attP2 (Control-RNAi), and P{TRiP.HMS01060}attP2 (Lon-RNAi2). The P{GD11336}-v21860 (PINK1-RNAi), P{KK101663}-v109629 (AFG3L2-RNAi1), and P{GD14030}-v36036 (Lon-RNAi1) RNAi lines were obtained from the Vienna Drosophila Resource Center. All other RNAi lines tested were obtained from the stock centers indicated in Table S1.

Thomas R.E., Andrews L.A., Burman J.L., Lin W.Y, & Pallanck L.J. (2014). PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix. PLoS Genetics, 10(5), e1004279.

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Drosophila Genetic Manipulation Protocols

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Flies were maintained on the standard cornmeal/soy flour/yeast fly food at 25 °C with a 12H/12H light and dark cycle. The following fly stocks were obtained for this study: da-Gal4 (Bloomington #8641); elav-Gal4 (Bloomington #8765); Mef2-Gal4 (Bloomington #27390); ey-Gal4 (Bloomington #5534 & #5535); r4-Gal4 (Bloomington #33832); pUASt-mitoGFP58 (link); pUASp-mCherry-Atg8a (Bloomington # 37750, from which Dr¹/TM3, Ser¹ was removed); pUASt-Atg1(6B) III (gift from Thomas Neufeld, University of Minnesota); Atg1 RNAi line (Bloomington #26731); Atg8a RNAi line (Vienna Drosophila RNAi Center #109654); Atg8a^KG07569/FM7c (Bloomington #14639); pUASt-Pink1 (ref. 38 (link)); pUASt-RNAi-Marf58 (link).
Constructs were injected at Rainbow Transgenic Flies, Inc. (http://www.rainbowgene.com). The constructs containing pIFM-Gal4 were inserted at the P{CaryP}attP1 site on the second chromosome (Bloomington #8621), while constructs with trans-activated genes were inserted at the P{CaryP}attP2 on the third chromosome (Bloomington #8622). Transgenic flies were balanced against the same background: w¹¹¹⁸; CyO/sna^Sco and w¹¹¹⁸; TM3, Sb¹/TM6B, Tb¹, respectively.

Kandul N.P., Zhang T., Hay B.A, & Guo M. (2016). Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila. Nature Communications, 7, 13100.

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Spiroplasma Infection in Drosophila Hemocytes

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Infected lines were generated in 2011 by injection of Spiroplasma-infected hemolymph in Oregon-R females. [17 (link)]. Infected lines have been maintained in the laboratory establishing genetically identical lines of Oregon-R Spiroplasma infected and uninfected. Hemocytes were observed on 4 weeks old females w1118; HmlΔGAL-4 > UAS-GFP [49 (link)]. For all the experiments, flies were maintained at 25 °C on standard cornmeal medium. Embryos were collected from 5 to 7 days old flies by using cages and yeasted grape juice plates. Lifespan experiments were done as described in [17 (link)]. The driver for REP assay (ey-GAL4) was obtained from Bloomington stock center (#8221). All experiments have been repeated three independent times.

Garcia-Arraez M.G., Masson F., Escobar J.C, & Lemaitre B. (2019). Functional analysis of RIP toxins from the Drosophila endosymbiont Spiroplasma poulsonii. BMC Microbiology, 19, 46.

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Transgenic Drosophila Eye Development

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Both transgenic lines GMR-hid¹⁰ (Grether et al., 1995 (link)) and GMR-hid³²⁶ (Fan and Bergmann, 2008 (link)) with insertions on the 2^nd and 3^rd chromosome, respectively, gave similar waves of cell death in developing eye discs. 4xGMR-rpr was obtained by combining CyO;2xGMR-rpr (Kurada and White, 1998 (link)) and homozygous GMR-rpr46 (White et al., 1996 (link)). GMR-hid^Ala5 (Bergmann et al., 1998 (link)), UAS-EGFRD^N (Freeman, 1996 (link)), UAS-rpr (Ryoo et al., 2002 ), th^33-1s (Goyal et al., 2000 (link)), GMR-diap1, GMR-BIR and th⁵ (Hay et al., 1995 (link)), GMR-p35 and UAS-p35 (Hay et al., 1994 ), uba-1^D6 (Lee et al., 2008 ), Cul3^gft2 and Cul1^[EX] (Ou et al., 2002 (link)) are as described. GMR-Gal4, ey-GAL4, elav^C155-GAL4, UAS-CD8GFP, ey-Flp, UAS-Flp, diap1-lacZ (th^j5c8) and Prosβ2 ^EP3067were obtained from the Bloomington Stock Center.

Fan Y, & Bergmann A. (2014). Multiple mechanisms modulate distinct cellular susceptibilities towards apoptosis in the developing Drosophila eye. Developmental cell, 30(1), 48-60.

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Drosophila Genetics: Allele and RNAi Strains

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The w¹¹¹⁸ allele (Bloomington Drosophila Stock Center no. 3605) was used as the wild-type control. The RNAi strains for BuGZ were obtained from the Vienna Drosophila RNAi library: w¹¹¹⁸; P{GD9485}v45449 (Vienna Drosophila Resource Center no. v45449). dsRNA-GD9485 (2L:16,744,705 … 16,745,060) was inserted in the third chromosome.
The UAS strains used were: UAS-upd transgenic fly generated through transformation of UAS-upd construct into W¹¹¹⁸ flies by standard methods (provided by X. Chen, Johns Hopkins University, Baltimore, MD; Kiger et al., 2001 (link)) and UAS-yki^{S111A.S168A.S250A.V5} (Bloomington Drosophila Stock Center no. 28817). The Gal4 allele used to drive the UAS lines is ey-Gal4 (Bloomington Drosophila Stock Center no. 5534), which is expressed in the Drosophila eyes.

Wan Y., Zheng X., Chen H., Guo Y., Jiang H., He X., Zhu X, & Zheng Y. (2015). Splicing function of mitotic regulators links R-loop–mediated DNA damage to tumor cell killing. The Journal of Cell Biology, 209(2), 235-246.

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Gal4 Drivers and Transgenic Fly Lines

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The Gal4 drivers, ey-Gal4 (5534) and AB1-Gal4 (1824) were obtained from Bloomington Drosophila Stock Center (BDSC). UAS-RFP (30556 on the second or 31417 on the third chromosome) (BDSC) was used for marking the Gal4 expression region. UAS-Tctp, UAS-Tctp RNAi, and UAS-hTCTP lines were as described [18 (link)], and another RNAi line (45532) was from Vienna Drosophila Resource Center (VDRC). For 14-3-3 knockdown, UAS-14-3-3ε RNAi (34884, BDSC and 31196R-3, National Institute of Genetics, Japan) and UAS-14-3-3ζ RNAi (VDRC v48725, BDSC 41878, and BDSC 28327) were used. UAS-myc-14-3-3ε was a gift from Dr. Efthimios Skoulakis. For double knockdown of Tctp and 14-3-3, ey > Tctp RNAi/CyO recombinants were crossed with 14-3-3ε RNAi or 14-3-3ζ RNAi lines. UAS-foxo^ORF (F000143, FLYORF) and UAS-foxo (9575, BDSC) were used for dFoxo overexpression. The cross of UAS-foxo (9575, BDSC) was carried out at 18 ^°C to reduce the severe phenotype of small salivary glands. All other flies were grown at 25^oC.

Nam S., Le T.P., Chung S, & Choi K.W. (2022). Tctp regulates the level and localization of Foxo for cell growth in Drosophila. Cell Death Discovery, 8, 146.

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