Uas lacz

Manufactured by Bloomington Drosophila Stock Center

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The UAS-lacZ is a genetic construct that contains the bacterial lacZ gene, which encodes the enzyme beta-galactosidase, under the control of the yeast Upstream Activating Sequence (UAS) promoter. This construct is commonly used as a reporter gene in Drosophila genetics research to visualize and analyze gene expression patterns.

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32 protocols using uas lacz

Drosophila Genetic Toolkit Protocol

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Flies were reared on a standard cornmeal and brown sugar medium at 25 °C unless otherwise indicated. The following strains were used in this work: ptc-Gal4, en-Gal4, nub-Gal4, GMR-Gal4, UAS-GFP [67 (link)], TRE-RFP [68 (link)], UAS-Egr, UAS-Hep [53 (link)], UAS-Puc, UAS-Bsk^DN [69 (link)], UAS-scrib-IR, UAS-P35 [34 (link)], yw hs-Flp; act > CD2 > Gal4 UAS-GFP [70 (link)], UAS-dTRAF2-IR, UAS-dTAK1-IR [14 (link)] and UAS-bsk-IR [71 (link)] were previously described. UAS-slik-IR (55,626), UAS-hep-IR (28,710), UAS-Hep^CA (6406), Df(2R)BSC603 (25,436), Df(2R)ED4065 (9069), Df(2R)ED4071 (24,117), UAS-LacZ (3956) and hs-Gal4 (1799) were obtained from the Bloomington stock center, UAS-egr-IR (45,253) was acquired from the Vienna Drosophila RNAi Center, UAS-slik-IR (4527R-3) was received from Japanese National Institute of Genetics (NIG), FRT42D slik^KG04837 (114,386) was obtained from Kyoto Stock Center, UAS-slik-IR (02634) was got from Tsing Hua Fly Center. yw hs-Flp; FRT42D ubi-GFP was provided by professor Haiyun Song, FRT42D was provided by professor Xianjue Ma, hs-Flp; FRT42D tub-Gal80; tub-Gal4 UAS-GFP was provided by professor Chenhui Wang. The human STK10 and Drosophila Slik expression plasmid, generated by PCR and subcloned into PUAST vector, was further used to produce the transgenic flies.

Li C., Zhu X., Sun X., Guo X., Li W., Chen P., Shidlovskii Y.V., Zhou Q, & Xue L. (2023). Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis. Cell Division, 18, 16.

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Drosophila PrP Expression Analysis

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The moderate UAS-HaPrP-M9 and strong UAS-HaPrP-M6 lines expressing hamster PrP and the motor neuron (BG380-Gal4) driver were described previously [5] (link). The reporter strains UAS-LacZ and UAS-CD8-GDP, and the ubiquitous da-Gal4 driver were obtained from the Bloomington Drosophila Stock Center. For expression of the PrP constructs, homozygous females for the Gal4 drivers were crossed with males bearing HaPrP-M9, HaPrP-M6, or LacZ transgenes. The crosses and their respective progenies were cultured at 25°C unless otherwise indicated.

Zhang Y., Casas-Tinto S., Rincon-Limas D.E, & Fernandez-Funez P. (2014). Combined Pharmacological Induction of Hsp70 Suppresses Prion Protein Neurotoxicity in Drosophila. PLoS ONE, 9(2), e88522.

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Genetic Manipulation of Drosophila Metabolism

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Virgin females carrying a daughterless (daGS)- or tubulin-GeneSwitch (tubGS) driver were crossed with (i) the following RNAi lines: 13131 (CG6020), 46799 (CG3683) 42162 (CG8905), 40466 (CG3731), 30892 (CG11015), 34664 (CG3612), (ii) UAS-lines carrying protein coding transgenes (NDI1, AOX, LacZ and ND-42-HA) or (iii) a wild type stock of Dahomey males [10 (link)]. Additionally, Dahomey virgin females were crossed with males carrying RNAi transgene against CG6020 as described above. Flies were collected following eclosion and transferred to new food for mating for 24 hours before being sorted for experiments. Mated 5 day old female flies maintained at 25°C were used for all experiments. Flies were maintained on standard media (1% agar, 1.5% sucrose, 3% glucose, 3.5% dried yeast, 1.5% maize, 1% wheat, 1% soya, 3% treacle, 0.5% propionic acid, 0.1% Nipagin) with a controlled 12hr:12hr light:dark cycle. All RNAi lines were obtained from the Vienna Drosophila Resource Center (VDRC) [5 (link)]. UAS-NDI1 and UAS-AOX have been previously described [11 (link)]. UAS-ND42-HA flies were a kind gift from the laboratory of Prof Hugo Bellen [12 (link)], UAS-LacZ was obtained from the Bloomington Drosophila Stock Center (BDSC) [13 (link)]. The daGS and tubGS drivers were a generous gift from the laboratories of Dr Veronique Monnier and Dr Scott Pletcher respectively.

Scialo F., Sriram A., Stefanatos R, & Sanz A. (2016). Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster. PLoS ONE, 11(8), e0161817.

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Drosophila Genetic Manipulations

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All stocks were raised on standard Drosophila media and crosses were performed at 25°C unless otherwise indicated. UAS-APLP1, UAS-APPL^sd were kindly provided by Dr. Merdes [22 (link)]; puc^H246, UAS-mmp1-IR, UAS-mmp2-IR, UAS-TIMP1, UAS-Ras^V12, UAS-Rho1-IR, UAS-p35, UAs-P53^DN were obtained from Bloomington Stock Center; sd-Gal4, UAS-Bsk^DN, UAS-Puc, UAS-Egr, UAS-Hep [70 (link)]; ptc-Gal4, en-Gal4, puc^{E69 34}, UAS-LacZ [71 (link)] were previously described.

Wang X., Sun Y., Han S., Wu C., Ma Y., Zhao Y., Shao Y., Chen Y., Kong L., Li W., Zhang F, & Xue L. (2017). Amyloid precursor like protein-1 promotes JNK-mediated cell migration in Drosophila. Oncotarget, 8(30), 49725-49734.

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Transgenic Fly Models of FUS Proteinopathy

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Tg flies expressing UAS-FUS wt or P525L were previously generated (28 (link)). The attB-attP40 expression system was used for the generation of tg flies expressing UAS-FUSwt (site-directed). gmr-GAL4, d42-GAL4, UAS-LacZ, UAS-Dco, UAS-Dco K38R, UAS-human CK1δ, UAS-Xenopus CK1ε kinase domain, UAS-human CK1δ (site-directed), UAS-human CK1α1 (site-directed) lines were purchased from Bloomington Drosophila Stock Center. Fly stocks were raised on standard Drosophila medium at 20 °C. Crosses between the Drosophila strains were carried out using standard procedures at 20 °C for the measurement of life span, 25 °C for other experiments.

Kishino Y., Matsukawa K., Matsumoto T., Miyazaki R., Wakabayashi T., Nonaka T., Kametani F., Hasegawa M., Hashimoto T, & Iwatsubo T. (2022). Casein kinase 1δ/ε phosphorylates fused in sarcoma (FUS) and ameliorates FUS-mediated neurodegeneration. The Journal of Biological Chemistry, 298(8), 102191.

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Drosophila Genetic Models for Parkinson's

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The following stocks were used for experiments: w¹¹¹⁸; UASp FL-clu, w¹¹¹⁸; UASp CLUH, clueless^d08713/CyO Act GFP, clueless^EP969/CyO KrGFP (Cox and Spradling, 2009 (link)), parkin¹/TM3 Act GFP (Cha et al., 2005 (link)), parkin²⁵/TM6B (Greene et al., 2003 (link)), w¹¹¹⁸; Mhc-GAL4, UAS-PINK1 RNAi (gift from Bingwei Lu, Stanford University, Palo Alto, CA), w¹¹¹⁸; UAS-parkin (gift from Leo J. Pallanck, University of Washington, Seattle, WA), w¹¹¹⁸; UAS-Atg1 (gift from Thomas Neufeld, University of Minnesota, Minneapolis, MN), Mhc-GAL4 (gift from Bingwei Lu), w¹¹¹⁸; UAS-PINK1 (gift from Bingwei Lu), and w¹¹¹⁸; park²⁵ (gift from L.J. Pallanck). Df(3)Pc-MK/TM2, PINK1^B9/FM7i (Park et al., 2006 (link)), Df(1)BSC535/FM7h, w¹¹¹⁸; UAS-lacZ, and Dp(1,3)DC026 were obtained from the Bloomington Drosophila Stock Center. UAS-parkin RNAi (VDRC^KK104363) UAS-Atg1 RNAi (VDRC^GD16133) and UAS-clu RNAi (VDRC^GD42138 and VDRC^KK100709) were all obtained from the Vienna Drosophila Resource Center. For wild type, y¹ w^67g23 was used. To obtain PINK1^B9/Df(1)BSC535 females, heterozygous PINK1^B9/FM7i virgins were crossed to Dp(1,3)DC026 males. PINK1^B9/Y; Dp(1,3)DC026/+ male progeny were then crossed to Df(1)BSC535/FM7h virgins. w⁻, non-FM7 females [PINK1^B9/Df(1)BSC535] were then dissected. Flies were reared on standard cornmeal fly media at 22°C or 25°C.

Sen A., Kalvakuri S., Bodmer R, & Cox R.T. (2015). Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila. Disease Models & Mechanisms, 8(6), 577-589.

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Genetic Modifier Screening in Drosophila

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All stocks used for our modifier screen were obtained from the Bloomington Drosophila Stock Center (Indiana, USA) and are listed in Results. The GMR-GAL4; UAS-cindr^RNAi2.21A/ SM5: TM6b line was generated from UAS-cindr^RNAi2.21A transgene [13 (link)] and the GMR-GAL4 driver line [17 (link)]. In addition, we utilized the following stocks: Canton-S, w¹¹¹⁸, UAS-lacZ and UAS-puc (gifts from R. Cagan), and bsk¹ (Bloomington stock number BL-3088), UAS-bsk (BL-9310), cbl^F165 (BL-9676), nopo^excl42 (BL-57335), nopo^Z1447 (BL-57334), puc^H246 (BL-4390), UAS-slpr^WT-HA (BL-58820), Traf4^EY09771 (BL-17600), UAS-Traf6.S (BL-58991) and Uev1a^DG14805 (BL-20440).

Ketosugbo K.F., Bushnell H.L, & Johnson R.I. (2017). A screen for E3 ubiquitination ligases that genetically interact with the adaptor protein Cindr during Drosophila eye patterning. PLoS ONE, 12(11), e0187571.

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Drosophila Genetics Protocols for Research

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All flies were raised on standard food at 25 °C. UAS-(GR)₈₀ and UAS-CONT-(GR)₈₀ flies as well as UAS-(G₄C₂)₁₆₀ and UAS-(G₄C₂)₅ flies have been obtained from earlier studies^{40 (link)}. A deficiency kit for the second chromosome and ELAV-GAL4, TubGAL80^ts, UAS-p35, lilli^k05431, lilli^A17-2, UAS-GFP, UAS-lacZ, OK³⁷¹-GAL4, and GMR-GAL4 fly stocks were from the Bloomington Drosophila Stock Center. Lilli RNAi line (v106142) was from the Vienna Drosophila Resource Center (VDRC). lilli^4U5 and UAS-lilli-HA fly stocks were a gift from Dr Ernst Hafen. The lilli^A17-2 allele was created by ethyl methanesulfonate mutagenesis. The nature of the mutation is unknown but is thought to be a loss of function allele^{55 (link)}. The lilli^k05431 allele is a P-element insertion^{56 (link)}. The lilli^4U5 allele has a short deletion resulting in a frameshift and a functional null allele^{56 (link)}.

Yuva-Aydemir Y., Almeida S., Krishnan G., Gendron T.F, & Gao F.B. (2019). Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD. Nature Communications, 10, 5466.

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Transgenic Drosophila Models of Neurodegenerative Diseases

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Drosophila were maintained on a 12:12 light/dark cycle on standard cornmeal-yeast agar medium at 25 °C. The following transgenic Drosophila strains were used in this study: UAS-FUS [19 (link)], UAS-TDP-43_TDPBR [55 (link)]. The GMR-Gal4, UAS-LacZ, UAS-CG42724^RNAi (stock #33737 and #55357) lines were obtained from Bloomington Stock Center. Detailed fly genotypes are listed in Additional file 2.

Pons M., Prieto S., Miguel L., Frebourg T., Campion D., Suñé C, & Lecourtois M. (2018). Identification of TCERG1 as a new genetic modulator of TDP-43 production in Drosophila. Acta Neuropathologica Communications, 6, 138.

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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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