Abstract:
: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 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 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. Environ. Mol. Mutagen. 59:18-29, 2018. © 2017 Wiley Periodicals, Inc.
journal_name
Environ Mol Mutagenjournal_title
Environmental and molecular mutagenesisauthors
Bemis JC,Avlasevich SL,Labash C,McKinzie P,Revollo J,Dobrovolsky VN,Dertinger SDdoi
10.1002/em.22154subject
Has Abstractpub_date
2018-01-01 00:00:00pages
18-29issue
1eissn
0893-6692issn
1098-2280journal_volume
59pub_type
杂志文章abstract::The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Test Procedures held a second harmonization meeting just prior to the U.S. Environmental Mutagen Society Meeting in New Orleans, LA, in April 2000. The discussion focused on several important aspects of the MLA, including: 1) cytoto...
journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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doi:10.1002/em.20436
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
doi:
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
doi:10.1002/em.21988
更新日期:2016-06-01 00:00:00
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
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更新日期:2016-03-01 00:00:00
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
doi:10.1002/em.10073
更新日期:2002-01-01 00:00:00
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
doi:10.1002/em.21724
更新日期:2012-10-01 00:00:00
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
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journal_title:Environmental and molecular mutagenesis
pub_type: 杂志文章
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