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Slicer Activity in Drosophila melanogaster S2 Extract

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Antiviral RNAi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 721))

Abstract

Reconstitution of RNA-inducing silencing complex (RISC) in vitro is a powerful biochemical technique to analyze crucial steps in RNA interference (RNAi) pathways. RISC contains an RNase enzyme, Argonaute, which is guided by small interfering RNA (siRNA) to recognize and silence its targets. Drosophila S2 cell extract is a good source of enzymes and factors that faithfully recapitulate essential steps in RISC assembly and function. In this chapter, we will describe how to prepare enzymatically active cell-free S2 extract to analyze the Slicer activity of RISC as well as the effects of viral RNAi suppressors which block this process.

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Acknowledgments

We thank Phillip Zamore, Benjamin Haley, Carla Saleh, and Ronald van Rij for their suggestions in preparing S2 extract. We thank Michel Tassetto from the Andino laboratory for testing this protocol. Thanks to Ashley Acevedo, Cecily Burrill, and Mark Borja for critical reading and valuable input while preparing the manuscript. Arabinda Nayak is a research fellow supported by N.I.H. grants (AI40085 and AI064738) to Raul Andino.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, University of California, San Francisco, CA, USA

    Raul Andino

Authors
  1. Arabinda Nayak
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  2. Raul Andino
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Correspondence to Raul Andino .

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Editors and Affiliations

  1. Inst. for Infection, Inflammation & Immu, Dept. Medical Microbiology, Radboud Univ.Nijmegen Medical Ctr, Geert Grooteplein 28, Nijmegen, 6525 GA, Netherlands

    Ronald P. van Rij

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© 2011 Humana Press

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Nayak, A., Andino, R. (2011). Slicer Activity in Drosophila melanogaster S2 Extract. In: van Rij, R. (eds) Antiviral RNAi. Methods in Molecular Biology, vol 721. Humana Press. https://doi.org/10.1007/978-1-61779-037-9_14

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  • DOI: https://doi.org/10.1007/978-1-61779-037-9_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-036-2

  • Online ISBN: 978-1-61779-037-9

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