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Detection of In Vivo Mutation in the Pig-a Gene of Mouse Bone Marrow Erythroids

  • Takafumi KimotoEmail author
  • Daishiro Miura
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1641)

Abstract

Detection of in vivo mutation is important for evaluating the health risks associated with chemicals. The Pig-a in vivo gene mutation assay has been developed over the last decade for this purpose. Most approaches for the assay, however, measure cells with a Pig-a mutant phenotype in erythrocytes from the peripheral blood, with the mutations causing the phenotype being difficult to determine directly. This chapter describes a procedure for detecting mutations in the Pig-a gene of phenotypically mutant mouse bone marrow erythroids, the precursors of peripheral blood erythrocytes. The strategy for molecular analysis of Pig-a gene mutation includes enrichment of GPI-anchor deficient cells with a cell sorter followed by a cloning and sequencing of Pig-a cDNAs.

Key words

CD24 GPI-anchor Flow cytometry Sorting Cloning 

Notes

Acknowledgments

The authors would like to thank Ms. Kumiko Suzuki-Okada and Xiao Mei Kobayashi for their cooperation and technical assistance with all the experiments.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Toxicology Research DepartmentTeijin Institute for Bio-medical Research, Teijin Pharma LimitedHinoJapan

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