Abstract
Cultured Drosophila cell lines have become an increasingly popular model system for cell biological and functional genomic studies. One of the most commonly used lines, S2 cells, is particularly useful as it is easy to grow and maintain in the lab, is highly susceptible to gene inhibition using RNAi and is well suited to high-resolution light microscopic assays. Here, we provide protocols for the routine culture and RNAi treatment of S2 cells and methods to prepare these cells for fluorescence microscopy. Using these techniques, loss-of-function experiments may be performed after 4–7 d of RNAi-mediated protein depletion.
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References
Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).
Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).
Echeverri, C.J. & Perrimon, N. High-throughput RNAi screening in cultured cells: a user's guide. Nat. Rev. Genet. 7, 373–384 (2006).
Schneider, I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27, 353–365 (1972).
Ramet, M. et al. Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416, 644–648 (2002).
Yanagawa, S. et al. Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling. J. Biol. Chem. 273, 32353–32359 (1998).
Rogers, S.L. et al. Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J. Cell Biol. 158, 873–884 (2002).
Rogers, S.L. et al. Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J. Cell Biol. 162, 1079–1088 (2003).
Saleh, M.C. et al. The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat. Cell Biol. 8, 793–802 (2006).
Ulvila, J. et al. Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells. J. Biol. Chem. 281, 14370–14375 (2006).
Ramadan, N. et al. Design and implementation of high-throughput RNAi screens in cultured Drosophila cells. Nat. Protoc. 2, 2245–2264 (2007).
Clemens, J.C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97, 6499–6503 (2000).
Echeverri, C.J. et al. Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat. Methods 3, 777–779 (2006).
Kulkarni, M.M. et al. Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat. Methods 3, 833–838 (2006).
Somma, M.P. et al. Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells. Mol. Biol. Cell 13, 2448–2460 (2002).
Bjorklund, M. et al. Identification of pathways regulating cell size and cell-cycle progression by RNAi. Nature 439, 1009–1013 (2006).
Magie, C.R. et al. Rho1 interacts with p120ctn and α-catenin, and regulates cadherin-based adherens junction components in Drosophila. Development 129, 3771–3782 (2002).
Drechsel, D.N. et al. A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos. Curr. Biol. 7, 12–23 (1997).
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Rogers, S., Rogers, G. Culture of Drosophila S2 cells and their use for RNAi-mediated loss-of-function studies and immunofluorescence microscopy. Nat Protoc 3, 606–611 (2008). https://doi.org/10.1038/nprot.2008.18
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DOI: https://doi.org/10.1038/nprot.2008.18
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