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Abstract

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.

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Acknowledgements

The design and construction of the first shRNAs were supported in part by the Janelia Farm Visitor Program. We thank G. Rubin, C. Zuker and T. Laverty for their interest and support; R. Hardy and C. Zuker for the data presented in Supplementary Table 2; B. Haley for helpful discussion on shRNAs; L. Cooley (Yale University) for the gift of the MTD-Gal4 line; and Z. Xuan for help with library design. S. Zusman and M. Tworoger of Genetic Services, Inc. generated the transgenic lines. R.Z. is supported by the Leukemia and Lymphoma Society. B.C. is supported by a PhD fellowship from the Boehringer Ingelheim Fonds. This work was supported by two US National Institute of General Medical Sciences R01 grants (GM067761 and GM084947) to N.P., an EU FP7 European Research Council starting grant to J.B. and contributions from the US National Institute of Neurological Disorders and Stroke.

Author information

Author notes

    • Jian-Quan Ni
    • , Rui Zhou
    •  & Lu-Ping Liu

    Present addresses: Gene Regulation Laboratory and Tsinghua Fly Center, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, China (J.-Q.N. and L.-P.L.) and Sanford-Burnham Medical Research Institute, La Jolla, California, USA (R.Z.).

    • Jian-Quan Ni
    • , Rui Zhou
    •  & Benjamin Czech

    These authors contributed equally to this work.

Affiliations

  1. Department of Genetics, Harvard Medical School, Howard Hughes Medical Institute, Boston, Massachusetts, USA.

    • Jian-Quan Ni
    • , Rui Zhou
    • , Lu-Ping Liu
    • , Laura Holderbaum
    • , Donghui Yang-Zhou
    • , Hye-Seok Shim
    • , Rong Tao
    • , Phillip Karpowicz
    • , Richard Binari
    • , Matthew Booker
    •  & Norbert Perrimon
  2. Watson School of Biological Sciences, Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

    • Benjamin Czech
    •  & Gregory J Hannon
  3. Institute of Molecular Biotechnology, Vienna, Austria.

    • Dominik Handler
    •  & Julius Brennecke
  4. Pediatric Surgical Research Labs, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Lizabeth A Perkins

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Contributions

J.-Q.N., R.Z. and B.C. carried out major experiments; L.-P.L., L.H., D.Y.-Z., H.-S.S., R.B., M.B. and L.A.P. produced the TRiP lines; P.K. performed the luciferase experiments in ovaries; D.H. and J.B. analyzed the piRNA pathway during oogenesis; and G.J.H. and N.P. supervised the project. R.Z., B.C., J.-Q.N., D.H., J.B., G.J.H. and N.P. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Norbert Perrimon.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Tables 1–3 and Supplementary Notes 1–3

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DOI

https://doi.org/10.1038/nmeth.1592

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