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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
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2102)

Abstract

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 

Notes

Acknowledgment

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

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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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