Abstract
RNA interference (RNAi) is a posttranscriptional gene silencing method that is triggered by double-stranded RNA (dsRNA). RNAi is used to inactivate genes of interest and provides a genetic tool for loss-of-function studies in a variety of organisms.
I have used this method to reveal the physiological roles of a number of endogenous proteins involved in mitochondrial DNA metabolism in Schneider cells, including the mitochondrial single-stranded DNA-binding protein. Here, I present experimental schemes of selective suppression of endogenous gene expression using RNAi in Drosophila Schneider S2 cells. With this method, the function of exogenous wild-type or mutant genes can be evaluated.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Wilson RC, Doudna JA (2013) Molecular mechanisms of RNA interference. Annu Rev Biophys 42:217–239. https://doi.org/10.1146/annurev-biophys-083012-130404
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811
Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404(6775):293–296
Celotto AM, Lee JW, Graveley BR (2005) Exon-specific RNA interference: a tool to determine the functional relevance of proteins encoded by alternatively spliced mRNAs. Methods Mol Biol 309:273–282. https://doi.org/10.1385/1-59259-935-4:273
Celotto AM, Graveley BR (2002) Exon-specific RNAi: a tool for dissecting the functional relevance of alternative splicing. RNA 8(6):718–724. https://doi.org/10.1017/s1355838202021064
Hutvagner G, Zamore PD (2002) RNAi: nature abhors a double-strand. Curr Opin Genet Dev 12(2):225–232. https://doi.org/10.1016/s0959-437x(02)00290-3
Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107(4):465–476. https://doi.org/10.1016/s0092-8674(01)00576-1
Farr CL, Matsushima Y, Lagina AT 3rd, Luo N, Kaguni LS (2004) Physiological and biochemical defects in functional interactions of mitochondrial DNA polymerase and DNA-binding mutants of single-stranded DNA-binding protein. J Biol Chem 279(17):17047–17053
Matsushima Y, Goto Y, Kaguni LS (2010) Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM). Proc Natl Acad Sci U S A 107(43):18410–18415. https://doi.org/10.1073/pnas.1008924107
Koelle MR, Talbot WS, Segraves WA, Bender MT, Cherbas P, Hogness DS (1991) The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67(1):59–77
Acknowledgments
I would like to thank Dr. Rebecca Porter from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 15H03096, 18H03180, and 18K19303.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Matsushima, Y. (2021). Selective Suppression of Endogenous Gene Expression Using RNAi in Drosophila Schneider S2 Cells. In: Oliveira, M.T. (eds) Single Stranded DNA Binding Proteins. Methods in Molecular Biology, vol 2281. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1290-3_19
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1290-3_19
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1289-7
Online ISBN: 978-1-0716-1290-3
eBook Packages: Springer Protocols