Detection of In Vivo Mutation in the Pig-a Gene of Mouse Bone Marrow Erythroids
Detection of in vivo mutation is important for evaluating the health risks associated with chemicals. The Pig-a in vivo gene mutation assay has been developed over the last decade for this purpose. Most approaches for the assay, however, measure cells with a Pig-a mutant phenotype in erythrocytes from the peripheral blood, with the mutations causing the phenotype being difficult to determine directly. This chapter describes a procedure for detecting mutations in the Pig-a gene of phenotypically mutant mouse bone marrow erythroids, the precursors of peripheral blood erythrocytes. The strategy for molecular analysis of Pig-a gene mutation includes enrichment of GPI-anchor deficient cells with a cell sorter followed by a cloning and sequencing of Pig-a cDNAs.
Key wordsCD24 GPI-anchor Flow cytometry Sorting Cloning
The authors would like to thank Ms. Kumiko Suzuki-Okada and Xiao Mei Kobayashi for their cooperation and technical assistance with all the experiments.
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