Uas dicer2

Manufactured by Bloomington Drosophila Stock Center

The UAS-Dicer2 is a genetic tool used in Drosophila research. It functions to increase the efficiency of RNA interference (RNAi) by expressing the Dicer-2 enzyme, which is responsible for cleaving double-stranded RNA into small interfering RNAs (siRNAs). This enhances the silencing of target genes in Drosophila models.

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14 protocols using uas dicer2

Drosophila Genetic Manipulation Protocol

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All flies were raised on glucose food (Archon) in a temperature- (25 °C) and humidity- (65%) controlled incubator with a 12-h light–dark cycle. 7- to 10-day-old males were used for all experiments, unless otherwise noted.
The following flies were used to manipulate inc and Cul3 as described previously^{41 (link),42 (link)}: UAS-inc-RNAi (VDRC stock #18225), elav^C155-Gal4, UAS-Dicer-2 (Bloomington stock #24651), UAS-Cul3-RNAi (NIG stock #11861R-2), along with the isogenic iso31 strain used for outcrossing. For neuronal Cul3 knockdown experiments, the UAS-Dicer-2 line (Bloomington stock #24651) was crossed into the elav^C155-Gal4 line. Parental controls used for experiments were obtained by crossing expression driver and RNAi construct lines to the outcrossed wild-type line for heterozygous controls, accounting for differences in phenotypes affected by genetic background. rutabaga mutants (rut¹) were acquired from David Schneider.

Tener S.J., Lin Z., Park S.J., Oraedu K., Ulgherait M., Van Beek E., Martínez-Muñiz A., Pantalia M., Gatto J.A., Volpi J., Stavropoulos N., Ja W.W., Canman J.C, & Shirasu-Hiza M. (2024). Neuronal knockdown of Cullin3 as a Drosophila model of autism spectrum disorder. Scientific Reports, 14, 1541.

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Genetic manipulation of Drosophila stocks

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Details on the generation of strat mutant alleles and transgenic lines (UASp-Strat, UASp-Strat-HA and UASp-hMss4) can be found in Supplemental Experimental Procedures. The following Drosophila stocks were also used: Crag^CJ101 (Denef et al., 2008 (link)); Pis¹ (Wang and Montell, 2006 (link)); Rab8¹ (Giagtzoglou et al., 2012 (link)); UASp-YFP-Rab8, UASp-YFP-Rab8T22N (Rab8DN), UASp-YFP-Rab8Q67L (Rab8CA), and UASp-YFP-Rab10Q68L (Rab10CA) (Zhang et al., 2007 (link)); YFP^MYC-Rab8 (Dunst et al., 2015 (link));. UAS-RNAi-Crag (TRIP line HMS00241); UAS-dicer2 and UAS-mCD8-RFP (Bloomington Stock Center); tj-Gal4 (a gift from D. Bilder); GR1-Gal4 (Goentoro et al., 2006 (link)). The RNAi lines for strat (v45715, v105730) and Rab8 (v28092) were from the Vienna Drosophila RNAi Center. The protein trap lines Vkg-GFP (CC00791) (Buszczak et al., 2007 (link)) and Pcan-GFP (ZCL1700) (Morin et al., 2001 (link)) were obtained from Flytrap. Follicle cell clones for Strat, Rab8, Crag and Pis were induced using the UAS-Flipase/FRT method (Harrison and Perrimon, 1993 (link)).

Devergne O., Sun G.H, & Schüpbach T. (2017). Stratum, a Homolog of the Human GEF Mss4, Partnered with Rab8, Controls the Basal Restriction of Basement Membrane Proteins in Polarized Epithelial Cells. Cell reports, 18(8), 1831-1839.

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Genetic Manipulation of Drosophila Larvae

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The following strains were used: w¹¹⁸ (control), pcol-Gal4;UAS-mCD8-GFP (pcol>GFP) [11 (link)], PG125 dome-gal4 (dome>) [29 (link)], Cg25C-GFP [30 (link)], hhF4fGFP [31 (link)], UAS-col RNAi [20 (link)], UAS-srp RNAi (GD12779 Vienna Drosophila RNAi Center), UAS-Myc and UAS-TCF^DN [20 (link)], UAS-reaper and UAS-Dicer2 (Bloomington Stock Center). Larvae were raised, in uncrowded conditions from eggs collected for 8 or 12 hours, at 27°C except for RNAi interference experiments which were performed at 29°C. UAS-Dicer2 was co-expressed with RNAi constructs. Larvae were developmentally staged at 29°C as follows: early third larval instar (EL3): 66H after egg laying (AEL); mid third larval instar (ML3): 85H AEL, late third larval instar (LL3): 105H AEL.

Oyallon J., Vanzo N., Krzemień J., Morin-Poulard I., Vincent A, & Crozatier M. (2016). Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis. PLoS ONE, 11(2), e0148978.

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Overexpressing PJA1 in Drosophila Eye

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Fly stocks and crosses were maintained at standard condition (25°C). Rh1-GAL4 was employed for overexpressing various transgenes (Xiong et al., 2012 (link)). An UAST-PJA1 transgenic fly was generated. PJA1 was digested from the pCMV-HA-PJA1 construct using Not1- and Kpn1-specific forward and reverse primers, respectively, and cloned into the pUAST plasmid vector (Brand and Perrimon, 1993 (link)). The plasmid clones were sent to Centre for Cellular and Molecular Platforms (CCAMP), Bangalore, subcloned into pUAST-attB, and then used for microinjection and targeted integration into the third chromosome. Transgenic flies were scored based on eye color marker, and the stocks were subsequently balanced. Rh1-GAL4 was used to drive the transgene, and the expression of PJA1 was confirmed by Western blot analysis. UAS-Hsap\MJD.tr-Q78 flies harboring the previously reported truncated ataxin-3 construct were obtained from the Bloomington Stock Centre (#8150). Genetic crosses were carried out to make stable stocks by combining this construct with UAST-PJA1 and UAS dicer2 (Bloomington #24644), respectively. Experiments were carried out by crossing Rh1 GAL4 flies to the respective genotypes. The progeny flies were sorted for the appropriate genotypes and were maintained at 25°C by regular transfer to a new vial of fly food. Retinal histology was carried out on 8–10- and 35–40-d-old flies.

Ghosh B., Karmakar S., Prasad M, & Mandal A.K. (2021). Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity. Molecular Biology of the Cell, 32(17), 1579-1593.

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Drosophila Bj1 RNAi Knockdown

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Bj1 TRiP RNAi [Flybase stock 0036067], mCherry TRiP RNAi in attP2, UAS-Dicer2, UAS-p35 (III), 1407-Gal4, y w, and UAS-NLS:GFP::lacZ were all from the Bloomington Drosophila Stock Center (BDSC); the strong hypomorphic Bj1^FF32 allele and the UAS-Bj1 stock were courtesy of Jim Skeath (Washington Univ.). RNAi experiments were done at 26–28°C. We also used FRT 2A, Df (3L) exel 7210 (http://www.drosdel.org.uk) and wor-Gal4 (Albertson et al., 2004 (link)). Larvae were dissected at the specified hours ALH (where time ALH is reported independent of temperature shifts) or as wandering third instar larvae. When assessing Bj1 alleles over a Bj1 deficiency, virgins were used from the deficiency line and males used from the Bj1 allele line.

Joy T., Hirono K, & Doe C.Q. (2014). The RanGEF Bj1 promotes Prospero nuclear export and neuroblast self-renewal. Developmental neurobiology, 75(5), 485-493.

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Drosophila Genetics for Neurodegeneration

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GAL4/UAS fly crosses were grown on a cornmeal-agar diet at 29 °C from the egg-laying to the adult stage. dDOR mutant and UAS-dDOR-F (“FENLL”) and UAS-dDOR-L (“Long”) lines were from A.A. Teleman^{32 (link)}, elav-GAL4^C155 (neuronal driver), mef2-GAL4 (muscle driver), CG-GAL4 (fat body driver), UAS-mCherry::Atg8a, UAS-GFP::Atg8a, dPINK1^B9, UAS-SNCA^A30P, UAS-dParkin_IR (TRiP line HMS01800), UAS-dPINK1_IR (TRiP line JF01203), UAS-Dicer2 and UAS-mCD8::GFP were from the Bloomington Drosophila Stock Center (BDSC), UAS-mtKeima was from M.J. Clague and A.J. Whitworth^{33 (link)}, and TH-GAL4 was from S. Birman. UAS-mit::dendra2 line stains mitochondrial matrix and is described in^{34 (link)}. UAS-dDOR::GFP line (C-terminal tag) was generated using the pUASp-GFP-W gateway vector from T. Murphy and inserted in the fly genome by random P-element transgenesis.

Dinh E., Rival T., Carrier A., Asfogo N., Corti O., Melon C., Salin P., Lortet S, & Kerkerian-Le Goff L. (2021). TP53INP1 exerts neuroprotection under ageing and Parkinson’s disease-related stress condition. Cell Death & Disease, 12(5), 460.

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Generating Transgenic Drosophila Lines

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Standard genetic methods were used to generate lines containing multiple transgenes. Transgenic lines used in this study include: UAS-Apc2-GFP and UAS-Dicer2 from the Bloomington Drosophila Stock Center, 221-Gal4 from Dr. Wes Grueber, For EB1 comet assays in da neurons, line containing UAS-Dicer2; 221-Gal4, UAS-EB1-GFP/TM6 was crossed with RNAi lines from the Vienna Drosophila RNAi Center (VDRC). The RNAi lines used as controls were Rtnl2 RNAi (#33320) and γ-tubulin37C (a maternal γ-tubulin not expressed in somatic cells (Wiese 2008 (link))) RNAi (#25271). We have previously validated these as controls (Chen et al. 2012 (link); Mattie et al. 2010 (link)). Other RNAi lines from VDRC were: Klp64D (#45373), Klp68D (#27943), Kap3 (#45400), and Apc (#51469). Larvae were grown on standard fly media consisting of cornmeal, yeast, dextrose, sucrose, and agar. Caps of food with fly embryos were collected every 24h and aged 3 days at 25°C. On the third day imaging experiments were performed.

Weiner A.T., Lanz M.C., Goetschius D.J., Hancock W.O, & Rolls M.M. (2016). Kinesin-2 and Apc function at dendrite branch points to resolve microtubule collisions. Cytoskeleton (Hoboken, N.J.), 73(1), 35-44.

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Genetic Screen Protocols for Drosophila

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For the genetic screens, flies were ordered from Tsinghua Fly Center, NIG-Fly, Vienna Drosophila RNAi Center, and FLYORF. The results of the screens are shown in Supplementary data S1. Tool strains used in this study: UAS-Dicer2 (a gift from T.Tabata); nos-Cas9 (34 (link)); vasa-GAL4 (35 (link)); balancers, w¹¹¹⁸, ZH-attP-86Fb, FRT42B, ubiGFP, FRT42B, FRT42D, ubiGFP, FRT42D, nos-GAL4, hs-FLP, nos-GAL4,UAS-FLP/TM6, Cre and da-Gal4 were ordered from Bloomington Drosophila Stock Center.
Cbc related flies: UAS-cbcRNAi (NIG#5970R-3) from NIG-Fly; UAS-cbcRNAi (v100686/v20998) from Vienna Drosophila RNAi Center. cbc^Q5stop and cbc^A37T from Bloomington Drosophila Stock Center; cbc^8fs5 was generated by CRISPR-Cas9 resulting in a 5 bp-deletion after the 8th amino acid. Cbc-HA was a genomicly tagged fly, in which 3 × HA was fused to cbc C terminus before stop codon by CRISPR-Cas9. Transgenic flies of UAS-Cbc, UAS-Cbc-flag, UAS-Cbc^A37T-flag, UAS-mCLP1 and UAS-hCLP1 were generated by inserting the pUASt-attB construct to attP landing site of ZH-attP-86Fb flies (36 (link)).

Wu J., Li X., Gao Z., Pang L., Liu X., Huang X., Wang Y, & Wang Z. (2021). RNA kinase CLP1/Cbc regulates meiosis initiation in spermatogenesis. Human Molecular Genetics, 30(17), 1569-1578.

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Drosophila Genetics and Experimental Tools

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D. melanogaster flies were reared on standard agar–cornmeal medium (standard food) at 25° under a 12/12 hr light/dark cycle. w¹¹¹⁸ was used as the wild-type (control) strain. UAS-Gclc#3 and UAS-Gclc#6 transgenic strains (Orr et al. 2005 (link)) were kindly gifted from William C. Orr (Southern Methodist University). phm-GAL4#22 (McBrayer et al. 2007 (link)) and DMef2-GAL4 (Ranganayakulu et al. 1996 (link)) were also kind gifts from Michael B. O’Connor (University of Minnesota) and Eric N. Olson (University of Texas Southwestern Medical Center at Dallas), respectively. ppl-GAL4 (Colombani et al. 2003 (link)) and byn-GAL4 (Iwaki and Lengyel 2002 (link)) were obtained from Hiroko Sano (Kurume University, Japan) and Ryutaro Murakami (Yamaguchi University, Japan), respectively. arm-GAL4 (Loncle et al. 2007 (link)), elav-GAL4 (Luo et al. 1994 (link)), tubP-GAL4 (Lee and Luo 1999 (link)), UAS-dicer2 (#24650), and hsFLP ovoD FRT19A (#23880) were provided by the Bloomington Drosophila Stock Center. p{GawB}NP3084 (Nehme et al. 2007 ) was provided by the Kyoto Stock Center. UAS-nobo-IR (#101884KK and #40316GD) and UAS-Gclc-IR (#108022KK and #33512GD) were obtained from the Vienna Drosophila RNAi center. y¹ v¹ nos-phiC31; attP40 and y² cho² v¹; attP40{nos-Cas9}/CyO (Kondo and Ueda 2013 (link)) were obtained from the National Institute of Genetics, Japan.

Enya S., Yamamoto C., Mizuno H., Esaki T., Lin H.K., Iga M., Morohashi K., Hirano Y., Kataoka H., Masujima T., Shimada-Niwa Y, & Niwa R. (2017). Dual Roles of Glutathione in Ecdysone Biosynthesis and Antioxidant Function During Larval Development in Drosophila. Genetics, 207(4), 1519-1532.

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Drosophila Genetic Manipulation Protocol

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D. melanogaster flies were reared on a standard agar-cornmeal medium at 25 or 17°C under a 12:12 h light/dark cycle. w¹¹¹⁸ served as a control strain. phm-GAL4#22 (a gift from Michael B. O’Connor, University of Minnesota, MN; McBrayer et al., 2007 (link); Yamanaka et al., 2013 (link)) was used as the strain to drive forced gene expression in the PG. UAS-dicer2 (#24650) was obtained from the Bloomington Drosophila Stock Center. UAS-pabp-IR (#22007) and UAS-mld-IR (#17329) were obtained from the Vienna Drosophila Resource Center. Transgenic RNAi experiments were conducted by crossing these UAS RNAi lines with w, UAS-dicer2, and phm-GAL4#22/TM6 Ubi-GFP. A strain carrying the 1.45 kb spok promoter-fused GFP cassette (spok>GFP) was previously described (Komura-Kawa et al., 2015 (link)).

Kamiyama T., Sun W., Tani N., Nakamura A, & Niwa R. (2020). Poly(A) Binding Protein Is Required for Nuclear Localization of the Ecdysteroidogenic Transcription Factor Molting Defective in the Prothoracic Gland of Drosophila melanogaster. Frontiers in Genetics, 11, 636.

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