In Vivo Rat T-Lymphocyte Pig-a Assay: Detection and Expansion of Cells Deficient in the GPI-Anchored CD48 Surface Marker for Analysis of Mutation in the Endogenous Pig-a Gene

  • Vasily N. DobrovolskyEmail author
  • Javier Revollo
  • Dayton M. Petibone
  • Robert H. Heflich
Part of the Methods in Molecular Biology book series (MIMB, volume 1641)


The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones. Subsequent sequencing of genomic DNA from the expanded clones confirms that the Pig-a assay detects exactly what it claims to detect—cells with mutations in the endogenous Pig-a gene. In addition, determining the spectra of Pig-a mutations provides information for better understanding the mutational mechanism of compounds of interest. Our methodology of combining phenotypic antibody labeling, magnetic enrichment, sorting, and single-cell clonal expansion can be used in genotoxicity/mutagenicity studies and in other general immunotoxicology research requiring identification, isolation, and expansion of extremely rare subpopulations of T-cells.

Key words

Glycosyl phosphatidylinositol Phosphatidylinositol glycan class A gene Flow cytometry Sorting Antibody 


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Vasily N. Dobrovolsky
    • 1
    Email author
  • Javier Revollo
    • 1
  • Dayton M. Petibone
    • 1
  • Robert H. Heflich
    • 1
  1. 1.Division of Genetic and Molecular ToxicologyNational Center for Toxicological Research, US Food and Drug AdministrationJeffersonUSA

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