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.
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.
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糖基磷脂酰肌醇 (GPI) 锚定蛋白缺陷可作为 Pig-a 基因突变的可靠报告者：来自基于 L5178Y/Tk /- 细胞和 CD90.2 抗原的体外测定的支持。
DOI：
10.1002/em.22154
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发表时间：
2018
影响因子：
2.8
通讯作者：
Dertinger,StephenD
中科院分区：
文献类型：
--
作者：
Bemis,JeffreyC;Avlasevich,SvetlanaL;Labash,Carson;McKinzie,Page;Revollo,Javier;Dobrovolsky,VasilyN;Dertinger,StephenD
Lack of cell surface glycosylphosphatidylinositol (GPI)‐anchored protein(s) has been used as a reporter ofPig‐agene mutation in several model systems. As an extension of this work, our laboratory initiated development of anin vitromutation 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 nonmutagenic compounds for 24 hr 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 nonmutagens 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 reflectsbona fide Pig‐agene 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 thePig‐agene. The continued investigation of L5178Y/Tk+/−cells, CD90.2 labeling, and flow cytometric analysis as the basis of anin vitromutation assay is clearly supported by this work. These data also provide evidence of the reliability of using GPI anchor‐deficiency as a valid reporter ofPig‐agene mutation. Environ. Mol. Mutagen. 59:18–29, 2018. © 2017 Wiley Periodicals, Inc.
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