Glycosylphosphatidylinositol (GPI) Anchored Protein Deficiency Serves as a Reliable Reporter of Pig-a Gene Mutation—Support from an In vitro Assay Based on L5178Y/Tk+/− Cells and the CD90.2 Antigen

摘要

Lack of cell surface glycosylphosphatidylinositol (GPI)-anchored protein(s) has been used as a reporter of Pig-a gene mutation in several model systems. As an extension of this work, our laboratory initiated development of an in vitro mutation assay based on the flow cytometric assessment of CD90.2 expression on the cell surface of the mouse lymphoma cell line L5178Y/Tk^+/−. Cells were exposed to mutagenic and non-mutagenic compounds for 24 hours followed by washout and incubation for an additional 7 days. Following this mutant manifestation time, cells were labeled with fluorescent antibodies against CD90.2 and CD45 antigens. These reagents indicated the presence of GPI-anchored proteins and general cell surface membrane receptor integrity, respectively. Instrument set-up was aided by parallel processing of a GPI anchor-deficient subclone. Results show that the mutagens reproducibly caused increased frequencies of mutant phenotype cells, while the non-mutagens did not. Further modifications to the method, including application of a viability dye and an isotype control for instrument set-up, were investigated. As a means to verify that the GPI-anchored protein-negative phenotype reflects bona fide Pig-a gene mutation, sequencing was performed on 38 CD90.2-negative L5178Y/Tk^+/− clones derived from cultures treated with ethyl methanesulfonate. All clones were found to have mutation(s) within the Pig-a gene. The continued investigation of L5178Y/Tk^+/− cells, CD90.2 labeling, and flow cytometric analysis as the basis of an in vitro mutation assay is clearly supported by this work. These data also provide evidence of the reliability of using GPI anchor-deficiency as a valid reporter of Pig-a gene mutation.