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Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies

Nature Protocols volume 3, pages 948954 (2008) | Download Citation

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Abstract

Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chemical and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, we provide a step-by-step protocol for a hybrid chemical-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the reduction of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR+ cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12–72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.

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Acknowledgements

We thank Justin Bosch for comments on the manuscript and help in testing the protocol for adult ablations, Stephen J. Johnson (Washington University) for his suggestion to use the NTR/Mtz system in zebrafish, Jeff Mumm and Eric Schroeter (Washington University) for providing the CFP-NTR construct and Ana Ayala and Koroboshka Brand for expert help maintaining the fish. R.M.A. was supported by a postdoctoral fellowship from the JDRF. This work was supported in part by grants from the NIH (NHLBI and NIDDK) and the Packard Foundation to D.Y.R.S.

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  1. Department of Biochemistry and Biophysics, Programs in Developmental Biology, Genetics and Human Genetics, Liver Center, Diabetes Center and the Cardiovascular Research Institute, University of California, San Francisco, 1550 Fourth Street, San Francisco, California 94158-2324, USA.

    • Silvia Curado
    • , Didier Y R Stainier
    •  & Ryan M Anderson

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Correspondence to Didier Y R Stainier.

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https://doi.org/10.1038/nprot.2008.58

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