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A high-throughput in vivo micronucleus assay for genome instability screening in mice

Nature Protocols volume 10, pages 205215 (2015) | Download Citation

Abstract

We describe a sensitive, robust, high-throughput method for quantifying the formation of micronuclei, markers of genome instability, in mouse erythrocytes. Micronuclei are whole chromosomes or chromosome segments that have been separated from the nucleus. Other methods of detection rely on labor-intensive, microscopy-based techniques. Here we describe a 2-d, 96-well plate–based flow cytometric method of micronucleus scoring that is simple enough for a research technician experienced in flow cytometry to perform. The assay detects low levels of genome instability that cannot be readily identified by classic phenotyping, using 25 μl of blood. By using this assay, we have screened >10,000 blood samples and discovered novel genes that contribute to vertebrate genome maintenance, as well as novel disease models and mechanisms of genome instability disorders. We discuss experimental design considerations, including statistical power calculation, we provide troubleshooting tips and we discuss factors that contribute to a false-positive increase in the number of micronucleated red blood cells and to experimental variability.

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Acknowledgements

We thank M. Hitcham and N. Harman for assistance with blood collections, W. Cheng for assistance with flow cytometry during high-throughput screening and K. Dry for comments on the manuscript. R.E.M. is supported by Cancer Research UK (CRUK; project grant C20510/A12401). D.J.A. is supported by CRUK. D.J.A. and B.L.N. are supported by the Wellcome Trust. Research in the Jackson Laboratory is funded by CRUK program grant no. C6/A11224, the European Research Council and the European Community Seventh Framework Programme grant agreement no. HEALTH-F2-2010-259893 (DDResponse). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. G.B. is funded by CRUK program grant no. C6/A11224.

Author information

Affiliations

  1. The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK.

    • Gabriel Balmus
    •  & Stephen P Jackson
  2. Maintenance of Genome Stability, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.

    • Gabriel Balmus
    •  & Stephen P Jackson
  3. The Sanger Mouse Genetics Project, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.

    • Natasha A Karp
  4. Cytometry Core Facility, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.

    • Bee Ling Ng
  5. Experimental Cancer Genetics, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.

    • David J Adams
    •  & Rebecca E McIntyre

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Contributions

D.J.A. and S.P.J. conceived the idea of screening mice from large-scale mouse phenotyping pipelines for micronucleated erythrocytes. G.B. and R.E.M. performed the experimental analysis and wrote the manuscript with comments from all authors. B.L.N. assisted with flow cytometry and N.A.K. performed statistical analysis.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to David J Adams or Rebecca E McIntyre.

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https://doi.org/10.1038/nprot.2015.010

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