Detection of Pig-a Mutant Erythrocytes in the Peripheral Blood of Rats and Mice

  • Vasily N. DobrovolskyEmail author
  • Xuefei Cao
  • Javed A. Bhalli
  • Robert H. Heflich
Part of the Methods in Molecular Biology book series (MIMB, volume 2102)


The endogenous X-linked phosphatidyl inositol glycan class A gene (Pig-a) can be used as a reporter of in vivo somatic cell mutation in rats and mice. Pig-a mutant cells are deficient in specific protein surface markers and can be identified and quantified by immunofluorescent staining followed by high-throughput flow cytometry. Pig-a mutation detection is commonly performed with red blood cells (RBCs) because: (1) the low volumes of blood required for determining mutant frequencies in RBCs allow multiple samplings on small laboratory animals over extended periods of time; (2) the execution of the RBC assay is easy and the interpretation of the results is straightforward; and (3) RBC Pig-a mutant frequencies are known within hours of sample collection. Two endpoints are determined in the assay: the frequency of mutant total RBCs and the frequency of mutant reticulocytes. When Pig-a mutation is used to assess the in vivo mutagenic potential of suspect hazards, the frequency of mutant reticulocytes is an early indicator of mutagenic potential, while the mutant frequency in total RBCs can be measured more rapidly and with greater precision.

Key words

Gene mutation Glycosyl phosphatidyl inositol Flow cytometry Reticulocytes CD59 surface marker CD24 surface marker Antibodies 



The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.


  1. 1.
    Ames BN, Durston WE, Yamasaki E, Lee FD (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci U S A 70:2281–2285CrossRefGoogle Scholar
  2. 2.
    Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10:789–799CrossRefGoogle Scholar
  3. 3.
    Cimino MC (2006) Comparative overview of current international strategies and guidelines for genetic toxicology testing for regulatory purposes. Environ Mol Mutagen 47:362–390CrossRefGoogle Scholar
  4. 4.
    Aidoo A, Morris SM, Casciano DA (1997) Development and utilization of the rat lymphocyte hprt mutation assay. Mutat Res 387:69–88CrossRefGoogle Scholar
  5. 5.
    Dobrovolsky VN, Casciano DA, Heflich RH (1999) Tk+/− mouse model for detecting in vivo mutation in an endogenous, autosomal gene. Mutat Res 423:125–136CrossRefGoogle Scholar
  6. 6.
    Jones IM, Burkhart-Schultz K, Carrano AV (1985) A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes. Mutat Res 147:97–105CrossRefGoogle Scholar
  7. 7.
    Stambrook PJ, Shao C, Stockelman M, Boivin G, Engle SJ, Tischfield JA (1996) APRT: a versatile in vivo resident reporter of local mutation and loss of heterozygosity. Environ Mol Mutagen 28:471–482CrossRefGoogle Scholar
  8. 8.
    Parsons BL, Heflich RH (1997) Genotypic selection methods for the direct analysis of point mutations. Mutat Res 387:97–121CrossRefGoogle Scholar
  9. 9.
    Dycaico MJ, Provost GS, Kretz PL, Ransom SL, Moores JC, Short JM (1994) The use of shuttle vectors for mutation analysis in transgenic mice and rats. Mutat Res 307:461–478CrossRefGoogle Scholar
  10. 10.
    Nohmi T, Masumura KI (2004) Gpt delta transgenic mouse: a novel approach for molecular dissection of deletion mutations in vivo. Adv Biophys 38:97–121CrossRefGoogle Scholar
  11. 11.
    Hayashi H, Kondo H, Masumura K, Shindo Y, Nohmi T (2003) Novel transgenic rat for in vivo genotoxicity assays using 6-thioguanine and Spi- selection. Environ Mol Mutagen 41:253–259CrossRefGoogle Scholar
  12. 12.
    Gossen JA, de Leeuw WJ, Tan CH et al (1989) Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo. Proc Natl Acad Sci U S A 86:7971–7975CrossRefGoogle Scholar
  13. 13.
    Dobrovolsky VN, Shaddock JG, Mittelstaedt RA et al (2009) Evaluation of Macaca mulatta as a model for genotoxicity studies. Mutat Res 673:21–28CrossRefGoogle Scholar
  14. 14.
    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 49:614–621CrossRefGoogle Scholar
  15. 15.
    Phonethepswath S, Bryce SM, Bemis JC, Dertinger SD (2008) Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential. Mutat Res 657:122–126CrossRefGoogle Scholar
  16. 16.
    Bryce SM, Bemis JC, Dertinger SD (2008) In vivo mutation assay based on the endogenous Pig-a locus. Environ Mol Mutagen 49:256–264CrossRefGoogle Scholar
  17. 17.
    Kinoshita T, Fujita M, Maeda Y (2008) Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: recent progress. J Biochem 144:287–294CrossRefGoogle Scholar
  18. 18.
    Miura D, Dobrovolsky VN, Kimoto T, Kasahara Y, Heflich RH (2009) Accumulation and persistence of Pig-A mutant peripheral red blood cells following treatment of rats with single and split doses of N-ethyl-N-nitrosourea. Mutat Res 677:86–92CrossRefGoogle Scholar
  19. 19.
    Dertinger SD, Phonethepswath S, Franklin D et al (2010) Integration of mutation and chromosomal damage endpoints into 28-day repeat dose toxicology studies. Toxicol Sci 115:401–411CrossRefGoogle Scholar
  20. 20.
    Dobrovolsky VN, Boctor SY, Twaddle NC et al (2010) Flow cytometric detection of Pig-A mutant red blood cells using an erythroid-specific antibody: application of the method for evaluating the in vivo genotoxicity of methylphenidate in adolescent rats. Environ Mol Mutagen 51:138–145CrossRefGoogle Scholar
  21. 21.
    Phonethepswath S, Franklin D, Torous DK et al (2010) Pig-a mutation: kinetics in rat erythrocytes following exposure to five prototypical mutagens. Toxicol Sci 114:59–70CrossRefGoogle Scholar
  22. 22.
    Dobrovolski VN, Elespuru RK, Bigger CA, Robison TW, Heflich RH (2011) Monitoring humans for somatic mutations in the endogenous pig-a gene using red blood cells. Environ Mol Mutagen 52:784–794CrossRefGoogle Scholar
  23. 23.
    Shemansky JM, McDaniel LP, Klimas C, Dertinger SD, Dobrovolsky VN, Kimoto T, Horibata K, Polli JE, Heflich RH (2019) Pig‐a gene mutation database. Environ Mol Mutagen 60(8):759–762CrossRefGoogle Scholar
  24. 24.
    Grant SG, Jensen RH (1993) Use of hematopoietic cells and markers for the detection and quantitation of human in vivo somatic mutation. In: Garratty G (ed) Immunobiology of transfusion medicine. Marcel Dekker, New York, pp 299–323Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Vasily N. Dobrovolsky
    • 1
    Email author
  • Xuefei Cao
    • 1
  • Javed A. Bhalli
    • 2
  • Robert H. Heflich
    • 1
  1. 1.Division of Genetic and Molecular ToxicologyNational Center for Toxicological ResearchJeffersonUSA
  2. 2.Toxicology/Safety AssessmentCovance Laboratories Inc.GreenfieldUSA

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