title: A high-throughput in vivo micronucleus assay for genome instability screening in mice.
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.
authors.affiliation: 1] The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK. [2] Maintenance of Genome Stability, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Balmus
authors.affiliation: The Sanger Mouse Genetics Project, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.
authors.forename: Natasha A
authors.initials: NA
authors.lastname: Karp
authors.affiliation: Cytometry Core Facility, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.
authors.forename: Bee Ling
authors.initials: BL
authors.lastname: Ng
authors.affiliation: 1] The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK. [2] Maintenance of Genome Stability, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Jackson
authors.affiliation: Experimental Cancer Genetics, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
authors.affiliation: Experimental Cancer Genetics, The Wellcome Trust Sanger Institute, Genome Campus, Cambridge, UK.
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: McIntyre
mesh_terms: D000818:Animals; D004912:Erythrocytes / Q000378:metabolism; D004920:Erythropoiesis / Q000235:genetics / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods; D042822:Genomic Instability / Q000235:genetics*; D057166:High-Throughput Screening Assays / Q000379:methods*; D051379:Mice; D015162:Micronucleus Tests / Q000379:methods*