Actin gal4

Manufactured by Bloomington Drosophila Stock Center

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The Actin-GAL4 is a transgenic Drosophila line that expresses the GAL4 transcription factor under the control of the Actin5C promoter. This line can be used to drive the expression of UAS-controlled transgenes in a wide range of Drosophila tissues and cell types.

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13 protocols using actin gal4

Drosophila Transgenic Line Generation

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The following fly stocks were used: Actin-GAL4 (Bloomington Drosophila Stock Center–BDSC), TH-GAL4 (BDSC), UAS-SNCA^A53T,^{69 (link)} and UAS-T7-DMiro-WT.^{70 (link)}UAS-T7-DMiro-KK were generated by injecting pUAST-T7-DMiro-KK into flies by Bestgene Inc (Chino Hills, CA). All fly stocks and experiments were kept at 25°C with a 12:12 h light:dark cycle and constant humidity (65%) on standard sugar-yeast-agar (SYA) medium (15 g/L agar, 50 g/L sugar, 100 g/L autolyzed yeast, 6 g/L nipagin, and 3 mL/L propionic acid).^{71 (link)} Flies were raised at standard density in 200 mL bottles unless otherwise stated. All fly lines were backcrossed 6 generations into a w¹¹¹⁸ background to ensure a homogeneous genetic background. The ages of flies for each experiment were stated in figures. Experiments were carried out on mated females.^{72 (link)}

Bharat V., Durairaj A.S., Vanhauwaert R., Li L., Muir C.M., Chandra S., Kwak C.S., Guen Y.L., Nandakishore P., Hsieh C.H., Rensi S.E., Altman R.B., Greicius M.D., Feng L, & Wang X. (2023). A mitochondrial inside-out iron-calcium signal reveals drug targets for Parkinson’s disease. Cell reports, 42(12), 113544.

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Transgenic Drosophila for SMS Protein Study

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Unless specified, flies were maintained on a cornmeal-molasses-yeast medium at 25 °C, 65% humidity, 12 h light/12 h dark. The following fly strains were used in the studies: actin-GAL4, elav-GAL4, UAS-mCherry-GFP-Atg8a, GstE1^EP2231 obtained from Bloomington Drosophila Stock Center; CG4300^e00382 obtained from the Exelixis Collection at the Harvard Medical School; dSms-GFP^CB04249 obtained from Dr Spradling’s lab (FlyTrap project^{62 (link)}). The following plasmids were generated to make the transgenic flies: pTFW-hSMS^wt, pTFW-hSMS⁴⁴³, pTFW-dSms^RA.

Li C., Brazill J.M., Liu S., Bello C., Zhu Y., Morimoto M., Cascio L., Pauly R., Diaz-Perez Z., Malicdan M.C., Wang H., Boccuto L., Schwartz C.E., Gahl W.A., Boerkoel C.F, & Zhai R.G. (2017). Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome. Nature Communications, 8, 1257.

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Drosophila Tau Transgenic Lines

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GMR-GAL4, elav-GAL4, actin-GAL4, tubulin-Gal4, tau^HM05101, tubP-GAL80^ts, UAS-Dcr-2, tau^Df(3R)MR22, Df(3R)BSC499, and UAS-hTau23 (UAS-MAPT.A59A) were obtained from the Bloomington Stock Center. UAS-dTau::GFP and tau^EP3597 were a gift of D. St. Johnston (University of Cambridge, Great Britain). Appl-Gal4 was obtained from Laura Torroja (Universidad Autonoma de Madrid, Spain). tau^GD25023 was received from the Vienna Drosophila RNAi Center (Vienna, Austria). olk¹ mutants are described in (Bettencourt da Cruz et al., 2005 (link)). Flies were raised under standard conditions at 25°C unless specifically described. To create the MAP60 and MAP205-expressing flies, we used LD02709 for MAP60 and LD12965, which were cloned into pUAST. Transgenic flies were generated by P-element transformation using Best Gene Inc. (Chino Hills, CA).

Bolkan B.J, & Kretzschmar D. (2014). Loss of Tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila. Developmental neurobiology, 74(12), 1210-1225.

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Drosophila RNAi and Overexpression Assays

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Canton S (Bloomington #1), Sgg RNAi (Bloomington #35364), Spalt-LacZ (gift from S. Newfeld), Actin-Gal4, Apterous-Gal4, HH-Gal4, MTD-Gal4 (Bloomington #31777), UAS-MWT-flag, UAS-MadA204/08-flag. All Drosophila crosses were carried out at room temperature 25°C.

Aleman A., Rios M., Juarez M., Lee D., Chen A, & Eivers E. (2014). Mad linker phosphorylations control the intensity and range of the BMP-activity gradient in developing Drosophila tissues. Scientific Reports, 4, 6927.

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Drosophila Kinetochore Mutant Analysis

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The P-element insertion mutant of mis12 (mis12^f03756), cenp-A mutant (cid^T11−2 and cid^T21−3), cenp-C mutant (cenp-C^prl41), cal1^c03646 and spc25 (spc25^A341) were examined as homozygotes unless otherwise noted. These lines, the elav-GAL4, OK6-Gal4, MHC-GAL4, Da-GAL4 and actin-GAL4 drivers, UAS-mCD8::GFP, and all other stocks were obtained from the Bloomington Drosophila Stock Center unless otherwise noted. Flies carrying the GFP-tagged kinetochore components, gEGFP-nuf2, gSpc25-EGFP, and gMis12-EGFP were generously provided by Dr. Christian Lehner (University of Zurich, Switzerland). All the RNAi Drosophila stocks (table S1) were from the Vienna Drosophila Resource Center (VDRC). Wild type refers to w¹¹¹⁸. All the fly stocks were maintained on standard cornmeal food at 25°C. The mixed sex of flies were used in this study.

Zhao G., Oztan A., Ye Y, & Schwarz T.L. (2019). Kinetochore Proteins Have a Post-Mitotic Function in Neurodevelopment. Developmental cell, 48(6), 873-882.e4.

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Drosophila Genetic Manipulations

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The UAS-DspastinRNAi Drosophila line used in this study was described previously [41 (link)]. UAS-Dspastin-myc and UAS-Dspastin^K467R-myc were generated by adding a myc epitope tag to the C-terminus of Dspastin and Dspastin^K467R constructs previously reported [41 (link)]. Six independent transgenic lines were derived for the K467R mutant and 5 for wild-type Dspastin. All lines were tested for protein expression by immunohistochemistry using different Gal4 driver lines. The two lines with the highest expression levels were chosen. The Gal4 activator lines Elav-Gal4, Mef2-Gal4, actin-Gal4, repo-Gal4 and the transgenic lines UAS-GFP-KDEL, UAS-GFP-Lamp and UAS-mCD8-GFP were obtained from the Bloomington Stock Center, Indiana University. Experimental crosses were performed at 28°C.

Papadopoulos C., Orso G., Mancuso G., Herholz M., Gumeni S., Tadepalle N., Jüngst C., Tzschichholz A., Schauss A., Höning S., Trifunovic A., Daga A, & Rugarli E.I. (2015). Spastin Binds to Lipid Droplets and Affects Lipid Metabolism. PLoS Genetics, 11(4), e1005149.

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COP9 Signalosome Regulation of Fly Growth

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‘Fly growth conditions’ were as published (9 (link)) with the following additions: Canton-S (CS) strain was used as wild type. RNAi-csn7 (#40691), RNAi-csn8 (#50565) and RNAi-csn2 (#48044) were obtained from VDRC stock center. Csn7^p (#18023), Actin-GAL4 (#3954) and Engrailed-GAL4 (#25752) were obtained from Bloomington stock center. Additional information including complementation analysis and transgenic flies is found in the Supplementary Methods.

Singer R., Atar S., Atias O., Oron E., Segal D., Hirsch J.A., Tuller T., Orian A, & Chamovitz D.A. (2014). Drosophila COP9 signalosome subunit 7 interacts with multiple genomic loci to regulate development. Nucleic Acids Research, 42(15), 9761-9770.

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Drosophila Fly Stocks for miRNA Studies

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The UAS-miRNA fly collection utilized in this study was previously described [103 (link)]. The following fly stocks were from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN, USA): w¹¹¹⁸, breathless-Gal4, engrailed-Gal4, dSRF-Gal4, actin-Gal4, UAS-GFP, UAS-white RNAi and miR-190^KO. The following stocks were from the Vienna Drosophila RNAi Center: UAS-fatiga RNAi (VDRC 103382), UAS-sima RNAi (VDRC 106504). The HRE-LacZ reporter [27 (link)], UAS-Fatiga B [30 (link)] and fga⁹/TM3 [29 (link)] lines were generated in our laboratory and previously described. The rhea^79a [40 (link)] mutant was kindly provided by Nicholas Brown.

De Lella Ezcurra A.L., Bertolin A.P., Kim K., Katz M.J., Gándara L., Misra T., Luschnig S., Perrimon N., Melani M, & Wappner P. (2016). miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga. PLoS Genetics, 12(5), e1006073.

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Drosophila Genetics: Gene Manipulation

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All control (wild-type) flies used in this study are yw. The following strains were used: plp²¹⁷² (Spradling et al., 1999 (link)), plp⁵ (Martinez-Campos et al., 2004 (link)), Df(3L)BrdR15 (stock 5354; Bloomington Drosophila Stock Center, Bloomington, IN), actin-Gal4 (stock 3954; Bloomington Drosophila Stock Center), CaM-TRiP line (TRiP.HMS01318; stock 34609; Bloomington, IN), FRT^42D,camⁿ³³⁹ (Heiman et al., 1996 (link)), and Ana1-tdTomato (Blachon et al., 2008 (link)). To generate the PLP wild-type (plp^WT) and mutant transgenes (plp^2KR, plp^2IQ), we first PCR amplified and directionally cloned full-length PLP^PF into pENTR/D vector for use in the Gateway cloning system. Site-directed mutagenesis was then conducted to generate plp^2KR and plp^2IQ. Each allele was recombined using Gateway reactions into the P-element destination vector pUWG (ubiquitin promoter, C-terminal GFP, DGRC). All transgenic flies were generated by BestGene (Chino Hills, CA) using standard P-element–mediated transformation.

Galletta B.J., Guillen R.X., Fagerstrom C.J., Brownlee C.W., Lerit D.A., Megraw T.L., Rogers G.C, & Rusan N.M. (2014). Drosophila pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies. Molecular Biology of the Cell, 25(18), 2682-2694.

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Drosophila Genetic Tools for OGA Study

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The 13618 OGA P element insertion, ogt/sxc mutants, Actin-Gal4, esg-GAL4, and the deficiency line spanning oga gene B9485 were from the Bloomington Stock Center. The UAS-OGA-RNAi fly line was obtained from VDRC [19 (link)]. The reported UAS-OGA overexpression lines were originally generated by Kaasik et al. [20 (link)]. The oga^del.1 mutant was generated by standard P-element excision protocol [21 ]. Flies were maintained at 25 °C in a humidified incubator. Drosophila MM media was purchased from KD medical (Columbia, MD, USA).

Akan I., Halim A., Vakhrushev S.Y., Clausen H, & Hanover J.A. (2021). Drosophila O-GlcNAcase Mutants Reveal an Expanded Glycoproteome and Novel Growth and Longevity Phenotypes. Cells, 10(5), 1026.

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