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pWormgatePro enables promoter-driven knockdown by hairpin RNA interference of muscle and neuronal gene products in Caenorhabditis elegans

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Invertebrate Neuroscience

Abstract

Recent advances in genome research and RNA interference (RNAi) technology have accelerated the adoption of genome-wide experimental approaches for determining gene function in the model organism Caenorhabditis elegans. Despite recent successes, the application of RNAi is limited when gene knockdown causes complex phenotypes or embryonic lethality. Recently, the high-throughput pWormgate cloning system has been introduced as a tool to efficiently generate heat-shock-inducible hairpin RNA constructs using the Gateway® recombination technology. We have modified pWormgate into a versatile hairpin cloning plasmid, pWormgatePro, which facilitates temporally and spatially inducible hairpin RNAi using constitutively active, tissue-specific promoters. To demonstrate its utility we knocked down unc-22 in body wall muscles as well as the axon guidance gene unc-5 in the nervous system indicating that promoter-driven hairpins can overcome the neuronal resistance to RNAi. Using pWormgatePro we also show that RNAi in the nervous system of C. elegans is non-autonomous and that spreading of the RNAi signal from neurons to muscle is substantially reduced but not abolished in spreading-defective sid-1 mutant animals. Our findings illustrate the effectiveness of pWormgatePro for gene silencing in muscle cells and neurons and bring forward the possibility of applying tissue-specific RNAi on a genome-wide scale.

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Abbreviations

dsRNA:

Double-stranded RNA

GFP:

Green fluorescent protein

hpRNA:

Hairpin RNA

IR:

Inverted repeat

ORF:

Open reading frame

RNAi:

RNA interference

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Acknowledgements

We thank the Caenorhabditis Genetics Center, Jonathan Hodgkin, Antonio Colavita and Joseph G. Culotti for providing strains. We would also like to thank Carolyn A. Behm and Stephen C. Trowell for sharing the pWormgate plasmids and Sarah Matthews for technical assistance. Michael Briese is supported by a Wellcome Trust 4 Year Doctoral Studentship in Neuroscience. Behrooz Esmaeili was supported by the MRC and is currently supported by the BBSRC.

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Authors and Affiliations

  1. MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, OX1 3QX, Oxford, UK

    Michael Briese, Behrooz Esmaeili & David B. Sattelle

  2. Genetics Unit, Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, Oxford, UK

    Behrooz Esmaeili

  3. School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, ACT 0200, Canberra, Australia

    Nicholas M. Johnson

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  1. Michael Briese
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  2. Behrooz Esmaeili
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  4. David B. Sattelle
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Correspondence to Michael Briese.

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Briese, M., Esmaeili, B., Johnson, N.M. et al. pWormgatePro enables promoter-driven knockdown by hairpin RNA interference of muscle and neuronal gene products in Caenorhabditis elegans . Invert Neurosci 6, 5–12 (2006). https://doi.org/10.1007/s10158-005-0011-x

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