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Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening

Nature Protocols volume 4, pages 1502–1512 (2009)Cite this article

Abstract

We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes ∼14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2–4 h.

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Figure 1: Outline of the experimental part of the protocol is schematized with the typical equipment used.
Figure 2: Primary cell cultures derived from Drosophila embryonic cells contain a mixture of cell populations including muscles and neurons.
Figure 3: Examples of RNAi phenotypes in primary muscle and neural cells.
Figure 4: Images of primary cells cultured on the human vitronectin-coated coverglass slides.
Figure 5

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Acknowledgements

We are very grateful for the technical support provided by the members at the Drosophila RNAi Screening Center, and R. Binari and members in the Perrimon lab. We thank J. Zirin for sharing his experience on primary-cell culture technique, and J. Zirin and R. Binari for critical comments on the manuscript. J.B. was supported by the Damon Runyon Cancer Research Foundation Fellowship DRG-1716-02. N.P. is an investigator of the Howard Hughes Medical Institute.

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Author notes

  1. Jianwu Bai and Katharine J Sepp: These authors contributed equally to this work.

Authors and Affiliations

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

    Jianwu Bai, Katharine J Sepp & Norbert Perrimon

  2. Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA

    Norbert Perrimon

Authors
  1. Jianwu Bai
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  2. Katharine J Sepp
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  3. Norbert Perrimon
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Contributions

J.B. and K.S. intellectually and experimentally contributed to this work; N.P. contributed to the reagents and analytical tools and was intellectually involved. J.B. wrote the paper. K.S. and N.P. edited the paper.

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Correspondence to Jianwu Bai.

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Bai, J., Sepp, K. & Perrimon, N. Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening. Nat Protoc 4, 1502–1512 (2009). https://doi.org/10.1038/nprot.2009.147

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