Abstract
Ribonucleic acid (RNA) interference triggered by double-stranded RNA has become a powerful tool for generating loss-of-function phenotypes. It is used to inactivate genes of interest and represents an elegant approach to genome functional analysis by reverse genetics. In Drosophila, RNA interference has been used in both cell culture and animals. We have adopted this approach to reveal the physiological roles of a number of proteins involved in mitochondrial deoxyribonucleic acid metabolism, and present here experimental schemes to induce the stable expression of double-stranded RNA in Schneider cells and in transgenic Drosophila.
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
Jorgensen, R. A. (2003) Sense cosuppression in plants: past, present and future, in RNAi: A Guide to Gene Silencing (Hannon, G. J., ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 5–22.
Cogoni, C., Irelan, J. T., Schumacher, M., Schmidhauser, T. J., Selker, E. U., and Macino, G. (1996) Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. EMBO J. 15, 3153–3163.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.
Cogoni, C. and Macino, G. (2000) Post-transcriptional gene silencing across kingdoms. Curr. Opin. Genet. Dev. 10, 638–643.
Tomari, Y. and Zamore, P. D. (2005) Perspective: machines for RNAi. Genes Dev. 19, 517–529.
Hammond, S. M., Bernstein, E., Beach, D., and Hannon, G. J. (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296.
Kennerdell, J. R. and Carthew, R. W. (1998) Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017–1026.
Koelle, M. R., Talbot, W. S., Segraves, W. A., Bender, M. T., Cherbas, P., and Hogness, D. S. (1991) The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67, 59–77.
Brand, A. H. and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.
Duffy, J. B. (2002) GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34, 1–15.
Spradling, A. C. (1986) P element-mediated transformation, in Drosophila a Practical Approach (Roberts, D. B., ed.), IRL Press, Oxford, UK, pp. 175–197.
Farr, C. L., Matsushima, Y., Lagina, A. T., 3rd, Luo, N., and Kaguni, L. S. (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, 17,047–17,053.
Lefai, E., Calleja, M., Ruiz de Mena, I., Lagina, A. T., 3rd, Kaguni, L. S., and Garesse, R. (2000) Overexpression of the catalytic subunit of DNA polymerase gamma results in depletion of mitochondrial DNA in Drosophila melanogaster. Mol. Gen. Genet. 264, 37–46.
Matsushima, Y., Adan, C., Garesse, R., and Kaguni, L. S. (2005) Drosophila mitochondrial transcription factor B1 modulates mitochondrial translation but not transcription or DNA copy number in Schneider cells. J. Biol. Chem. 280, 16,815–16,820.
Matsushima, Y., Garesse, R., and Kaguni, L. S. (2004) Drosophila mitochondrial transcription factor B2 regulates mitochondrial DNA copy number and transcription in Schneider cells. J. Biol. Chem. 279, 26,900–26,905.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Matsushima, Y., Adán, C., Garesse, R., Kaguni, L.S. (2007). Functional Analysis by Inducible RNA Interference in Drosophila melanogaster . In: Leister, D., Herrmann, J.M. (eds) Mitochondria. Methods in Molecular Biology™, vol 372. Humana Press. https://doi.org/10.1007/978-1-59745-365-3_15
Download citation
DOI: https://doi.org/10.1007/978-1-59745-365-3_15
Publisher Name: Humana Press
Print ISBN: 978-1-58829-667-2
Online ISBN: 978-1-59745-365-3
eBook Packages: Springer Protocols