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

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


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 



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


  1. 1.
    Dobrovolsky VN, Miura D, Heflich RH, Dertinger SD (2010) The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment. Environ Mol Mutagen 51:825–835CrossRefPubMedGoogle Scholar
  2. 2.
    Gollapudi BB, Lynch AM, Heflich RH, Dertinger SD, Dobrovolsky VN, Froetschl R, Horibata K, Kenyon MO, Kimoto T, Lovell DP, Stankowski LF Jr, White PA, Witt KL, Tanir JY (2015) The in vivo Pig-a assay: a report of the international workshop on genotoxicity testing (IWGT) workgroup. Mutat Res 783:23–35CrossRefGoogle Scholar
  3. 3.
    Heflich RH, Dobrovolsky VN, Dertinger SD (2015) Continued progress in developing the Pig-a gene mutation assay. Mutagenesis 30:313–314CrossRefPubMedGoogle Scholar
  4. 4.
    Miura D (2014) The in vivo Pig-a gene mutation assay. Genes Environ 36:169–173CrossRefGoogle Scholar
  5. 5.
    Phonethepswath S, Bryce SM, Bemis JC, Dertinger SD (2008) Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential. Mutat Res 657:122–126CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Miura D, Dobrovolsky VN, Kasahara Y, Katsuura Y, Heflich RH (2008) Development of an in vivo gene mutation assay using the endogenous Pig-a gene: I. Flow cytometric detection of CD59-negative peripheral red blood cells and CD48-negative spleen T-cells from the rat. Environ Mol Mutagen 49:614–621CrossRefPubMedGoogle Scholar
  7. 7.
    Bryce SM, Bemis JC, Dertinger SD (2008) In vivo mutation assay based on the endogenous Pig-a locus. Environ Mol Mutagen 49:256–264CrossRefPubMedGoogle Scholar
  8. 8.
    Miura D, Dobrovolsky VN, Mittelstaedt RA, Kasahara Y, Katsuura Y, Heflich RH (2008) Development of an in vivo gene mutation assay using the endogenous pig-a gene: II. Selection of Pig-a mutant rat spleen T-cells with proaerolysin and sequencing Pig-a cDNA from the mutants. Environ Mol Mutagen 49:622–630CrossRefPubMedGoogle Scholar
  9. 9.
    Kimoto T, Suzuki K, Kobayashi XM, Dobrovolsky VN, Heflich RH, Miura D, Kasahara Y (2011) Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression. Mutat Res 723:36–42CrossRefPubMedGoogle Scholar

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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