Establishing a novel Pig‐a gene mutation assay in L5178YTk+/− mouse lymphoma cells

The X-linked Pig-a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig-a mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig-a mutation in various in vivo assays. Here, we describe an in vitro Pig-a mutation assay in L5178YTk+/– mouse lymphoma cells, in which mutant-phenotype cells are measured by flow cytometry using a fluorescent anti-CD90 antibody. Increased frequencies of CD90-deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate, and 7,12-dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting-dilution cloning assay using proaerolysin selection. Individual CD90-deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig-a gene. Pig-a mutations with agent-specific signatures were found in nearly all clones that developed from sorted CD90-deficient cells. These results indicate that a Pig-a mutation assay can be successfully conducted in L5178YTk+/− cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig-a assays. Environ. Mol. Mutagen., 2017. Published 2017. This article is a US Government work and is in the public domain in the USA.