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

  • Vasily N. Dobrovolsky
  • Xuefei Cao
  • Javed A. Bhalli
  • Robert H. Heflich
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1105)

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 

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Copyright information

© Humana Press 2014

Authors and Affiliations

  • Vasily N. Dobrovolsky
    • 1
  • Xuefei Cao
    • 2
  • Javed A. Bhalli
    • 2
  • Robert H. Heflich
    • 2
  1. 1.Division of Genetic and MolecularU.S. FDA/NCTRJeffersonUSA
  2. 2.Division of Genetic and Reproductive ToxicologyNational Center for Toxicological ResearchJeffersonUSA

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