RNAi Screening in Drosophila Cells and in Vivo

Overview

Journal Methods

Specialty Biochemistry

Date 2014 Mar 1

PMID 24576618

Citations 22

Authors

Stephanie E Mohr

Affiliations

Soon will be listed here.

Abstract

Here, I discuss how RNAi screening can be used effectively to uncover gene function. Specifically, I discuss the types of high-throughput assays that can be done in Drosophila cells and in vivo, RNAi reagent design and available reagent collections, automated screen pipelines, analysis of screen results, and approaches to RNAi results verification.

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References

Wiles A, Ravi D, Bhavani S, Bishop A . An analysis of normalization methods for Drosophila RNAi genomic screens and development of a robust validation scheme. J Biomol Screen. 2008; 13(8):777-84. PMC: 2956424. DOI: 10.1177/1087057108323125. View

Seinen E, Burgerhof J, Jansen R, Sibon O . RNAi experiments in D. melanogaster: solutions to the overlooked problem of off-targets shared by independent dsRNAs. PLoS One. 2010; 5(10). PMC: 2948504. DOI: 10.1371/journal.pone.0013119. View

Steinbrink S, Boutros M . RNAi screening in cultured Drosophila cells. Methods Mol Biol. 2008; 420:139-53. DOI: 10.1007/978-1-59745-583-1_8. View

Echeverri C, Beachy P, Baum B, Boutros M, Buchholz F, Chanda S . Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods. 2006; 3(10):777-9. DOI: 10.1038/nmeth1006-777. View

Agrawal T, Sadaf S, Hasan G . A genetic RNAi screen for IP₃/Ca²⁺ coupled GPCRs in Drosophila identifies the PdfR as a regulator of insect flight. PLoS Genet. 2013; 9(10):e1003849. PMC: 3789835. DOI: 10.1371/journal.pgen.1003849. View

Marinho J, Martins T, Neto M, Casares F, Pereira P . The nucleolar protein Viriato/Nol12 is required for the growth and differentiation progression activities of the Dpp pathway during Drosophila eye development. Dev Biol. 2013; 377(1):154-65. DOI: 10.1016/j.ydbio.2013.02.003. View

Mohr S, Perrimon N . RNAi screening: new approaches, understandings, and organisms. Wiley Interdiscip Rev RNA. 2011; 3(2):145-58. PMC: 3249004. DOI: 10.1002/wrna.110. View

Jenett A, Rubin G, Ngo T, Shepherd D, Murphy C, Dionne H . A GAL4-driver line resource for Drosophila neurobiology. Cell Rep. 2012; 2(4):991-1001. PMC: 3515021. DOI: 10.1016/j.celrep.2012.09.011. View

Zhang , Chung , OLDENBURG . A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen. 2000; 4(2):67-73. DOI: 10.1177/108705719900400206. View

10.

Cherbas L, Gong L . Cell lines. Methods. 2014; 68(1):74-81. PMC: 4048767. DOI: 10.1016/j.ymeth.2014.01.006. View

11.

Fisher K, Wright V, Taylor A, Zeidler M, Brown S . Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway. BMC Genomics. 2012; 13:506. PMC: 3526451. DOI: 10.1186/1471-2164-13-506. View

12.

Lyne R, Smith R, Rutherford K, Wakeling M, Varley A, Guillier F . FlyMine: an integrated database for Drosophila and Anopheles genomics. Genome Biol. 2007; 8(7):R129. PMC: 2323218. DOI: 10.1186/gb-2007-8-7-r129. View

13.

Perrimon N, Friedman A, Mathey-Prevot B, Eggert U . Drug-target identification in Drosophila cells: combining high-throughout RNAi and small-molecule screens. Drug Discov Today. 2007; 12(1-2):28-33. DOI: 10.1016/j.drudis.2006.10.006. View

14.

Cherry S . Genomic RNAi screening in Drosophila S2 cells: what have we learned about host-pathogen interactions?. Curr Opin Microbiol. 2008; 11(3):262-70. PMC: 2525573. DOI: 10.1016/j.mib.2008.05.007. View

15.

Dietzl G, Chen D, Schnorrer F, Su K, Barinova Y, Fellner M . A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature. 2007; 448(7150):151-6. DOI: 10.1038/nature05954. View

16.

Huang D, Sherman B, Lempicki R . Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4(1):44-57. DOI: 10.1038/nprot.2008.211. View

17.

Flockhart I, Booker M, Hu Y, McElvany B, Gilly Q, Mathey-Prevot B . FlyRNAi.org--the database of the Drosophila RNAi screening center: 2012 update. Nucleic Acids Res. 2011; 40(Database issue):D715-9. PMC: 3245182. DOI: 10.1093/nar/gkr953. View

18.

Kaplow I, Singh R, Friedman A, Bakal C, Perrimon N, Berger B . RNAiCut: automated detection of significant genes from functional genomic screens. Nat Methods. 2009; 6(7):476-7. DOI: 10.1038/nmeth0709-476. View

19.

Baum B, Cherbas L . Drosophila cell lines as model systems and as an experimental tool. Methods Mol Biol. 2008; 420:391-424. DOI: 10.1007/978-1-59745-583-1_25. View

20.

Read R, Fenton T, Gomez G, Wykosky J, VandenBerg S, Babic I . A kinome-wide RNAi screen in Drosophila Glia reveals that the RIO kinases mediate cell proliferation and survival through TORC2-Akt signaling in glioblastoma. PLoS Genet. 2013; 9(2):e1003253. PMC: 3573097. DOI: 10.1371/journal.pgen.1003253. View