The In Vivo Pig-a Gene Mutation Assay

The phosphatidylinositol glycan anchor biosynthesis, Class A gene (Pig-a in rodents, PIG-A in humans) codes for a catalytic subunit of the N-acetylglucosamine transferase complex that is involved in an early step of glycosylphosphatidyl inositol (GPI) anchor synthesis, and GPI anchors tether specific protein markers to the surface of various types of cells. Two distinct strategies for Pig-a gene mutation assay used peripheral blood have been developed, one using flow cytometry and the other using limiting-dilution cloning. The limiting-dilution cloning assay using bacterial protoxin, proaerolysin, as a selective agent is resource intensive. For routine analysis of mutant frequency, the flow cytometric procedures employing fluorescently labeled antibodies against GPI-anchored markers (e.g., anti-CD59 for rat red blood cells, anti-CD24 for mouse red blood cells) are preferred for routine analysis of mutant frequency. The advantage of the cloning assay, however, is in-depth analyses of the mutant genotype (mutational spectra analysis). The characteristics of the flow cytometric Pig-a gene mutation assays, i.e., induced Pig-a mutant frequencies were persistence and the effect of split doses of chemicals were additive, make the assay on reticulocytes and total red blood cells an attractive possibility for developing detailed mutagenicity data in vivo. Although a large amount of information must be gathered on assay performance, progress toward the goal in the last few years has been rapid, and multi-laboratory trial has been initiated and ongoing.