Uas mcd8 rfp

Manufactured by Bloomington Drosophila Stock Center

Sourced in France, United States

The UAS-mCD8::RFP is a genetic construct that expresses the membrane-bound red fluorescent protein (mCD8::RFP) under the control of the UAS (Upstream Activating Sequence) promoter. It is commonly used as a reporter to visualize and label specific cell populations in Drosophila genetic experiments.

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10 protocols using uas mcd8 rfp

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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Detecting γ-tubulin in Drosophila BO

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γ-tubulin 37C-GFP (Bloomington #56831) was used to detect γ-tubulin. The wild-type line y¹¹¹⁸ (Bloomington #5905) was utilized for the immunohistochemical labeling experiments. Rh5-Gal4 (Mollereau et al., 2000 (link)) and UAS-mCD8::RFP (Bloomington #32219) were used to study the expression of the Rh5 in the BO.

Hartenstein V., Yuan M., Younossi-Hartenstein A., Karandikar A., Bernardo-Garcia F.J., Sprecher S, & Knust E. (2019). Serial Electron Microscopic Reconstruction of the Drosophila Larval Eye: Photoreceptors with a Rudimentary Rhabdomere of Microvillar-Like Processes. Developmental biology, 453(1), 56-67.

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Drosophila Genetic Manipulation Protocol

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All stock flies were raised at 25 °C on standard food. Crosses were performed using a standard procedure. The UAS-mCD8-RFP and UAS-mitoGFP lines were obtained from the Bloomington Drosophila Stock Center. The podocyte-specific driver snsGCN-Gal4 line was provided by Dr. Ross L. Cagan (University of Pennsylvania, Philadelphia, PA, USA).

Kim K., Cha S.J., Choi H.J., Kang J.S, & Lee E.Y. (2021). Dysfunction of Mitochondrial Dynamics in Drosophila Model of Diabetic Nephropathy. Life, 11(1), 67.

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Drosophila Fly Strains and Rearing

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Orco^Gal4 flies were generously provided by G. Galizia (University of Konstanz, Germany), lush^Gal4 flies (originally from R Benton, Université de Lausanne, Switzerland) from J-F. Ferveur (CSGA, Dijon, France) and elav^LexA, LexAOP-mCD8::GFP, UAS-mCD8::RFP and Est6 null mutant flies from the Bloomington Stock Center (stocks 52676, 32203, 27392 and 4211 respectively). All flies were raised at 25 °C on standard yeast/cornmeal/agar medium in a 12-hr light/12-hr dark cycle, 50–60% relative humidity.

Younus F., Fraser N.J., Coppin C.W., Liu J.W., Correy G.J., Chertemps T., Pandey G., Maïbèche M., Jackson C.J, & Oakeshott J.G. (2017). Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila. Scientific Reports, 7, 46188.

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Drosophila Transgenic Fly Stocks

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The following fly stocks were used: UAS-HPV16 E5, E6, E7 (generated in this study), UAS-hUBE3A, GMR-Gal4, UAS-p35, UAS-mCD8RFP, and CS were from Bloomington Drosophila stock center.

Hashemi L., Ormsbee M.E., Patel P.J., Nielson J.A., Ahlander J, & Padash Barmchi M. (2022). A Drosophila model of HPV16-induced cancer reveals conserved disease mechanism. PLOS ONE, 17(12), e0278058.

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Drosophila Genetic Manipulation Protocols

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Fly stocks were maintained on standard cornmeal-yeast-agar medium at 25°C, and on a 12/12 hr light/dark cycle. The MiMIC and CRIMIC flies were created in the Bellen lab (see Flypush or Supplementary file 2). UAS-2xEGFP, hs-Cre,vas-dϕC31, Trojan T2A-GAL4 triplet flies were from Dr. Ben White (Diao et al., 2015 (link)). The RMCE conversion of MiMICs with GFSTF and T2A-GAL4 cassettes was described in previous studies (Diao et al., 2015 (link); Nagarkar-Jaiswal et al., 2015a (link); Nagarkar-Jaiswal et al., 2015b (link)). The crossing schemes for CRIMICs are shown in Supplemental Information 1. btl-GAL4, Ilp2-GAL4, repo-GAL4, gcm-GAL4, UAS-mCD8::GFP, UAS-mCD8::RFP, P[acman] flies, and UAS-FLP flies were obtained from the Bloomington Drosophila Stock Center (BDSC, USA). UAS-if was from Dr. Celeste Berg (Beumer et al., 1999 (link)). NP1243-GAL4, NP2222-GAL4, and NP6250-GAL4 are from Kyoto Stock Center (Kyoto DGGR, Japan). Dfs flies were from BDSC or Kyoto DGGR. UAS-cDNA flies were from BDSC or FlyORF (Switzerland). y,w;attP40(y+){nos-Cas9(v+)}/CyO (Kondo and Ueda, 2013 (link)) and y,w;+/+; attP2(y+){nos-Cas9(v+)} (Ren et al., 2013 (link)) were isogenized in this work. See Supplementary file 2 for the genotypes and stock numbers of fly stocks. All references to FlyBase are based on FlyBase 2.0/FB2017_06 (Gramates et al., 2017 (link)).

Lee P.T., Zirin J., Kanca O., Lin W.W., Schulze K.L., Li-Kroeger D., Tao R., Devereaux C., Hu Y., Chung V., Fang Y., He Y., Pan H., Ge M., Zuo Z., Housden B.E., Mohr S.E., Yamamoto S., Levis R.W., Spradling A.C., Perrimon N, & Bellen H.J. (2018). A gene-specific T2A-GAL4 library for Drosophila. eLife, 7, e35574.

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Drosophila Genetic Manipulation Protocol

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Flies were kept on a cornmeal and agar medium at 25 °C according to standard protocols. Drosophila strains used include: sna¹ (25127), sna¹⁸ (3299), UAS-sna-IR^B (28679), UAS-mGFP (32197), bsk¹ (3088), UAS-Bsk, UAS-dFoxO (9575), Ser-GAL4 (6791), Sco/CyO (2555), UAS-mCD8-RFP (32219), UAS-dsh-IR (31306), wor¹(3155), wor⁴ (25170) and the deficiency kit were obtained from Bloomington Drosophila stock center. UAS-sna-IR^V (6263), UAS-puc-IR (3018), UAS-dlg-IR (41136), and UAS-wg-IR^V (13352) were obtained from Vienna Drosophila RNAi center. UAS-wg-IR^N (4889R-4), UAS-esg-IR^N-1 (3758R-1), and UAS-esg-IR^N-2 (3758R-5) were received from Fly Stocks of National Institute of Genetics (NIG-FLY). UAS-GFP-IR (0355) and UAS-wor-IR (GL00186) were obtained from TsingHua Fly Center. GMR-GAL4^{53 (link)}; Sd-GAL4 and ptc-GAL4^{39 (link)}; UAS-Egr, UAS-Egr^W, and UAS-Hid^{14 (link)}; UAS-Bsk^DN, UAS-Puc, and puc^E69^{42 (link)}; sev-GAL4, UAS-dTAK1, UAS-Hep^CA, UAS-Hep^WT, UAS-LacZ, pnr-GAL4, and UAS-GFP^{22 (link),29 (link),54 (link)}; dFoxO^Δ94 and UAS-dFoxO-IR^{39 (link)} were previously described. UAS-Sna^74b fly was a kind gift of J. Kumar. For all fly cross experiments, healthy unmated male and female parents were randomly assigned to different groups. Double-blinded method was employed during the experiments.

Wu C., Li Z., Ding X., Guo X., Sun Y., Wang X., Hu Y., Li T., La X., Li J., Li J.A., Li W, & Xue L. (2019). Snail modulates JNK-mediated cell death in Drosophila. Cell Death & Disease, 10(12), 893.

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Culturing and Experimental Handling of Drosophila and Platynereis

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Drosophila melanogaster stocks were cultured at 25 °C in a 12:12 h light–dark cycle, and we fed them with cornmeal medium (which was supplemented with molasses, fructose, and yeast). We used Canton-S as a wild-type strain (courtesy of R. Stocker), glass-Gal4 (courtesy of S. Kim) [38 (link)] and UAS-mCD8::RFP (Bloomington Stock Center, no. 32219).
Our wild-type P. dumerilii were a mixed population of worms, originally captured in the sea in Naples (Italy) and Arcachon (France). We also used r-opsin1-GFP worms (courtesy of F. Raible) [14 (link)]. These animals were kept in sea water at 22 °C, in a 16:8 h light–dark cycle. We maintained them synchronised to an artificial moon cycle, induced by slightly increasing the light intensity at night for 1 week per month (using a 10-W light bulb, to simulate the full moon). Platynereis had a varied diet that included Artemia salina, Tetraselmis marina, fish food, and spinach leaves. For our experiments (i.e. in situ hybridisation and microinjections), we crossed males and females and collected the fertilised eggs, as previously described [39 ]. The larvae that hatched from these eggs were kept at 18 °C.

Bernardo-Garcia F.J., Syed M., Jékely G, & Sprecher S.G. (2019). Glass confers rhabdomeric photoreceptor identity in Drosophila, but not across all metazoans. EvoDevo, 10, 4.

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Drosophila Genetics and Transgenic Techniques

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Flies were reared at 25°C on standard Drosophila yeast-cornmeal molasses media. The following stocks were used: OK107-Gal4, Sema-1a^k13702, Df(2l)Exel7039, PlexB^KG00878, PlexA^{EY 16548}, UAS-lacZ.btau.YES, UAS-mCD8-Gfp, UAS-mCD8-Rfp (Bloomington stock center, Bloomington, IN, USA), Sema-1a^CA7125 and PlexA^{Y D0269} (A. Spradling, Carnegie Institution for Science, Baltimore, MD, USA), 442-Gal4 (T. Préat, Ecole Supérieure de Physique et Chimie Industrielle, Paris, France), UAS-Sema-1a-RNAi, UAS-PlexB-RNAi and UAS-PlexA-RNAi (L. Luo, Stanford University, Stanford, CA, USA; The efficacy and specificity of these RNAi constructs has been previously reported (Hu et al., 2001 (link); Sweeney et al., 2007 (link); Yu et al., 2010 (link)), UAS-PlexB and UAS-Sema-1a (C. Goodman, University of California, Berkeley, CA, USA), UAS-Sema-1a^Δcyt1 (T. Godenschwege, University of Massachusetts, Amherst, MA, USA), UAS-Sema-1a^Δcyt2 (A. Kolodkin, Johns Hopkins University, Baltimore, MD, USA). For mosaic analysis with a repressible cell marker (MARCM) analyses, hsFLP; Sema-1a^k13702 FRT40a/FRT40A-Gal80; Tub-Gal4, UAS-Gfp-cd8 flies were heat shocked at 37°C for 1 h at the relevant developmental stage (Lee et al., 1999 (link)).

Zwarts L., Goossens T., Clements J., Kang Y.Y, & Callaerts P. (2016). Axon Branch-Specific Semaphorin-1a Signaling in Drosophila Mushroom Body Development. Frontiers in Cellular Neuroscience, 10, 210.

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Drosophila Mitochondrial Dynamics Protocols

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The following fly stocks were used: Tubulin-GAL4, Actin-GAL4, elav-GAL4, da-GAL4, UAS-mitoGFP, UAS-mCD8RFP (Bloomington Drosophila Stock Center, BDSC), dMIC60 LL02849 (Drosophila Genomics Resource Center, DGRC, Kyoto) (Schuldiner et al., 2008) , PINK1 PE704 (Clark et al., 2006) , PINK1 5 (Clark et al., 2006) , PINK1 RV (Park et al., 2006) , PINK1 B9 (Park et al., 2006) , Park 25 (Greene et al., 2003) , Park rva (Greene et al., 2003) , UAS-ND42 (Pogson et al., 2014) , UAS-Sicily (Pogson et al., 2014) , UAS-Drp1 (Poole et al., 2008) , UAS-MUL1 (Yun et al., 2014) . UAS-hPINK1 WT -Flag, UAS-hPINK1 DMTS -Flag, UAS-dMIC60 WT -Myc (Tsai et al., 2017) , UAS-dMIC60 T507A,T561A -Myc, UAS-hMIC60 WT -Myc, UAS-hMIC60 A4V -Myc, UAS-hMIC60 T11A -Myc, UAS-hMIC60 C17F -Myc, UAS-hMIC60 R25H -Myc, and UAS-hMIC60 R31C -Myc were generated using PhiC31 integrase-mediated transgenesis, with an insertion at an estimated position of 25C6 at the attP40 site (BestGene) (Markstein et al., 2008) . The age or developmental stage of flies for each experiment was stated in the Figure Legends. Male flies were used for PINK1 null flies because male PINK1 null flies are infertile and PINK1 is on the X chromosome, and an equal number of both male and female flies were used for the other genotypes.

Tsai P.I., Lin C.H., Hsieh C.H., Papakyrikos A.M., Kim M.J., Napolioni V., Schoor C., Couthouis J., Wu R.M., Wszolek Z.K., Winter D., Greicius M.D., Ross O.A, & Wang X. (2018). PINK1 Phosphorylates MIC60/Mitofilin to Control Structural Plasticity of Mitochondrial Crista Junctions. Molecular cell, 69(5).

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