Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl- N-nitrosourea: Direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression
► We developed a flow cytometric Pig-a assay for measuring gene mutation in the mouse. ► We used this assay to elucidate the generation of mutations in the Pig-a assay. ► The kinetics of Pig-a mutant manifestation is more rapid in bone marrow than in peripheral blood. ► CD24-negative erythroids have...
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creator | Kimoto, Takafumi Suzuki, Kumiko Kobayashi, Xiao mei Dobrovolsky, Vasily N. Heflich, Robert H. Miura, Daishiro Kasahara, Yoshinori |
description | ► We developed a flow cytometric
Pig-a assay for measuring gene mutation in the mouse. ► We used this assay to elucidate the generation of mutations in the
Pig-a assay. ► The kinetics of
Pig-a mutant manifestation is more rapid in bone marrow than in peripheral blood. ► CD24-negative erythroids have mutation in the
Pig-a gene. ► The
Pig-a assay on red blood cells is useful for the detection of
in vivo mutagenicity.
Our previous rat studies indicate that the endogenous
Pig-a gene is a promising reporter of
in vivo mutation and potentially useful as the basis for an
in vivo genotoxicity assay. The function of the Pig-a protein in the synthesis of glycosylphosphatidyl inositol (GPI) anchors is conserved in variety of eukaryotic cells, including human and rodent cells, which implies that
Pig-a mutants can be measured in a similar manner in different mammalian species. In the present study, we developed a flow cytometric
Pig-a assay for rapidly measuring gene mutation in the mouse. An antibody to TER-119, a specific cell-surface marker of murine erythroid lineage, was used to identify erythrocytes in peripheral blood (PB) and erythroids in bone marrow (BM). An antibody to CD24, a GPI-anchored protein, was used to identify
Pig-a mutants as CD24-negative cells. CD-1 mice were administered a single dose of 100
mg/kg
N-ethyl-
N-nitrosourea (ENU), and PB and BM were collected at 1, 2, and 4 weeks after dosing. While the
Pig-a mutant frequency (MF) in PB was increased moderately at 2 and 4 weeks after ENU dosing, the
Pig-a MF in BM was strongly increased starting at 1 week after the dosing, with the elevated MF persisting for at least 4 weeks after the dosing. We also used flow cytometric sorting to isolate CD24-negative erythroids from the BM of ENU-treated mice. cDNA sequencing indicated that these cells have mutations in the
Pig-a gene, with base-pair substitutions typical of ENU-induced mutation spectra. The results indicate that the
Pig-a mutation assay can be adapted for measuring mutation in BM erythroids and PB of mice. Taken together, the data suggest that
Pig-a mutants are fixed in the BM, where they further proliferate and differentiate; erythrocytes derived from these BM
Pig-a mutants transit from the BM and accumulate in PB. |
doi_str_mv | 10.1016/j.mrgentox.2011.03.016 |
format | Article |
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Pig-a assay for measuring gene mutation in the mouse. ► We used this assay to elucidate the generation of mutations in the
Pig-a assay. ► The kinetics of
Pig-a mutant manifestation is more rapid in bone marrow than in peripheral blood. ► CD24-negative erythroids have mutation in the
Pig-a gene. ► The
Pig-a assay on red blood cells is useful for the detection of
in vivo mutagenicity.
Our previous rat studies indicate that the endogenous
Pig-a gene is a promising reporter of
in vivo mutation and potentially useful as the basis for an
in vivo genotoxicity assay. The function of the Pig-a protein in the synthesis of glycosylphosphatidyl inositol (GPI) anchors is conserved in variety of eukaryotic cells, including human and rodent cells, which implies that
Pig-a mutants can be measured in a similar manner in different mammalian species. In the present study, we developed a flow cytometric
Pig-a assay for rapidly measuring gene mutation in the mouse. An antibody to TER-119, a specific cell-surface marker of murine erythroid lineage, was used to identify erythrocytes in peripheral blood (PB) and erythroids in bone marrow (BM). An antibody to CD24, a GPI-anchored protein, was used to identify
Pig-a mutants as CD24-negative cells. CD-1 mice were administered a single dose of 100
mg/kg
N-ethyl-
N-nitrosourea (ENU), and PB and BM were collected at 1, 2, and 4 weeks after dosing. While the
Pig-a mutant frequency (MF) in PB was increased moderately at 2 and 4 weeks after ENU dosing, the
Pig-a MF in BM was strongly increased starting at 1 week after the dosing, with the elevated MF persisting for at least 4 weeks after the dosing. We also used flow cytometric sorting to isolate CD24-negative erythroids from the BM of ENU-treated mice. cDNA sequencing indicated that these cells have mutations in the
Pig-a gene, with base-pair substitutions typical of ENU-induced mutation spectra. The results indicate that the
Pig-a mutation assay can be adapted for measuring mutation in BM erythroids and PB of mice. Taken together, the data suggest that
Pig-a mutants are fixed in the BM, where they further proliferate and differentiate; erythrocytes derived from these BM
Pig-a mutants transit from the BM and accumulate in PB.</description><identifier>ISSN: 1383-5718</identifier><identifier>ISSN: 0027-5107</identifier><identifier>EISSN: 1879-3592</identifier><identifier>DOI: 10.1016/j.mrgentox.2011.03.016</identifier><identifier>PMID: 21549855</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Animals ; Antibodies ; Biological and medical sciences ; Bone Marrow Cells - drug effects ; CD24 ; Erythrocytes ; Erythrocytes - drug effects ; Ethylnitrosourea - toxicity ; Flow cytometry ; Fundamental and applied biological sciences. Psychology ; Gene expression ; Gene mutation ; Genetics of eukaryotes. Biological and molecular evolution ; Glycosylphosphatidyl inositol ; Glycosylphosphatidylinositols - metabolism ; Medical sciences ; Membrane Proteins - genetics ; Mice ; Mutagenicity Tests - methods ; Mutation ; Phosphatidylinositol glycan complementation class A gene ; Protein synthesis ; Rodents ; Sorting ; Time Factors ; Toxicology</subject><ispartof>Mutation research, 2011-07, Vol.723 (1), p.36-42</ispartof><rights>2011 Elsevier B.V.</rights><rights>2015 INIST-CNRS</rights><rights>Copyright © 2011 Elsevier B.V. All rights reserved.</rights><rights>Copyright Elsevier BV Jul 14, 2011</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c456t-100f107710da6cca65119721ee7928425e43f27710d2c1e2c948a746c1d932853</citedby><cites>FETCH-LOGICAL-c456t-100f107710da6cca65119721ee7928425e43f27710d2c1e2c948a746c1d932853</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.mrgentox.2011.03.016$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=24289822$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21549855$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kimoto, Takafumi</creatorcontrib><creatorcontrib>Suzuki, Kumiko</creatorcontrib><creatorcontrib>Kobayashi, Xiao mei</creatorcontrib><creatorcontrib>Dobrovolsky, Vasily N.</creatorcontrib><creatorcontrib>Heflich, Robert H.</creatorcontrib><creatorcontrib>Miura, Daishiro</creatorcontrib><creatorcontrib>Kasahara, Yoshinori</creatorcontrib><title>Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl- N-nitrosourea: Direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression</title><title>Mutation research</title><addtitle>Mutat Res</addtitle><description>► We developed a flow cytometric
Pig-a assay for measuring gene mutation in the mouse. ► We used this assay to elucidate the generation of mutations in the
Pig-a assay. ► The kinetics of
Pig-a mutant manifestation is more rapid in bone marrow than in peripheral blood. ► CD24-negative erythroids have mutation in the
Pig-a gene. ► The
Pig-a assay on red blood cells is useful for the detection of
in vivo mutagenicity.
Our previous rat studies indicate that the endogenous
Pig-a gene is a promising reporter of
in vivo mutation and potentially useful as the basis for an
in vivo genotoxicity assay. The function of the Pig-a protein in the synthesis of glycosylphosphatidyl inositol (GPI) anchors is conserved in variety of eukaryotic cells, including human and rodent cells, which implies that
Pig-a mutants can be measured in a similar manner in different mammalian species. In the present study, we developed a flow cytometric
Pig-a assay for rapidly measuring gene mutation in the mouse. An antibody to TER-119, a specific cell-surface marker of murine erythroid lineage, was used to identify erythrocytes in peripheral blood (PB) and erythroids in bone marrow (BM). An antibody to CD24, a GPI-anchored protein, was used to identify
Pig-a mutants as CD24-negative cells. CD-1 mice were administered a single dose of 100
mg/kg
N-ethyl-
N-nitrosourea (ENU), and PB and BM were collected at 1, 2, and 4 weeks after dosing. While the
Pig-a mutant frequency (MF) in PB was increased moderately at 2 and 4 weeks after ENU dosing, the
Pig-a MF in BM was strongly increased starting at 1 week after the dosing, with the elevated MF persisting for at least 4 weeks after the dosing. We also used flow cytometric sorting to isolate CD24-negative erythroids from the BM of ENU-treated mice. cDNA sequencing indicated that these cells have mutations in the
Pig-a gene, with base-pair substitutions typical of ENU-induced mutation spectra. The results indicate that the
Pig-a mutation assay can be adapted for measuring mutation in BM erythroids and PB of mice. Taken together, the data suggest that
Pig-a mutants are fixed in the BM, where they further proliferate and differentiate; erythrocytes derived from these BM
Pig-a mutants transit from the BM and accumulate in PB.</description><subject>Animals</subject><subject>Antibodies</subject><subject>Biological and medical sciences</subject><subject>Bone Marrow Cells - drug effects</subject><subject>CD24</subject><subject>Erythrocytes</subject><subject>Erythrocytes - drug effects</subject><subject>Ethylnitrosourea - toxicity</subject><subject>Flow cytometry</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene expression</subject><subject>Gene mutation</subject><subject>Genetics of eukaryotes. Biological and molecular evolution</subject><subject>Glycosylphosphatidyl inositol</subject><subject>Glycosylphosphatidylinositols - metabolism</subject><subject>Medical sciences</subject><subject>Membrane Proteins - genetics</subject><subject>Mice</subject><subject>Mutagenicity Tests - methods</subject><subject>Mutation</subject><subject>Phosphatidylinositol glycan complementation class A gene</subject><subject>Protein synthesis</subject><subject>Rodents</subject><subject>Sorting</subject><subject>Time Factors</subject><subject>Toxicology</subject><issn>1383-5718</issn><issn>0027-5107</issn><issn>1879-3592</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkk1v1DAQhiMEoqXwFyoLCXHK4o98OJyoWloqldIDnC2vM9l4ldjB9rbdX8zf6Gx3lwoucPLI84z9zjuTZceMzhhl1YflbAwLcMnfzzhlbEbFDK-fZYdM1k0uyoY_x1hIkZc1kwfZqxiXlHIqqHyZHXBWFo0sy8Ps11ftbAcx6WS9I74jN3aRazKuknaJzL0DMuoQ_B2BsE598LaNRLuWTBDs1EPQA5kP3rf7vFkniMQ6MloDJAXQCVpyZ1NPrnNI_XrIMXA2BR_9CtMfyZkNYBKJ8HMFzli3eNJhzq5PSBf8-IcUA8MQiYMFqr4F0vlALm4uc-1M7wP-NgWfACXA_RQgRuzsdfai00OEN7vzKPtx_vn76Zf86tvF5enJVW6Ksko5o7RjtK4ZbXVljK5KxpqaM4C64bLgJRSi4495bhhw0xRS10VlWNsILktxlL3fvosSsJuY1GjjRq524FdRyaZhVUXFf5A1xSEVvELy7V_kEo1z2AZCFcoqCoFQtYUM-hoDdGoKFu1aK0bVZmXUUu1XRm1WRlGh8BoLj3evr-YjtL_L9juCwLsdoKPRQxfQZhufuILLRnKO3KctB-jvrYWgorE4T2gfx6tab_-l5QEsjOY5</recordid><startdate>20110714</startdate><enddate>20110714</enddate><creator>Kimoto, Takafumi</creator><creator>Suzuki, Kumiko</creator><creator>Kobayashi, Xiao mei</creator><creator>Dobrovolsky, Vasily N.</creator><creator>Heflich, Robert H.</creator><creator>Miura, Daishiro</creator><creator>Kasahara, Yoshinori</creator><general>Elsevier B.V</general><general>Elsevier</general><general>Elsevier BV</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7ST</scope><scope>7TM</scope><scope>7U7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>SOI</scope><scope>7X8</scope></search><sort><creationdate>20110714</creationdate><title>Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl- N-nitrosourea: Direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression</title><author>Kimoto, Takafumi ; Suzuki, Kumiko ; Kobayashi, Xiao mei ; Dobrovolsky, Vasily N. ; Heflich, Robert H. ; Miura, Daishiro ; Kasahara, Yoshinori</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c456t-100f107710da6cca65119721ee7928425e43f27710d2c1e2c948a746c1d932853</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Animals</topic><topic>Antibodies</topic><topic>Biological and medical sciences</topic><topic>Bone Marrow Cells - drug effects</topic><topic>CD24</topic><topic>Erythrocytes</topic><topic>Erythrocytes - drug effects</topic><topic>Ethylnitrosourea - toxicity</topic><topic>Flow cytometry</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene expression</topic><topic>Gene mutation</topic><topic>Genetics of eukaryotes. Biological and molecular evolution</topic><topic>Glycosylphosphatidyl inositol</topic><topic>Glycosylphosphatidylinositols - metabolism</topic><topic>Medical sciences</topic><topic>Membrane Proteins - genetics</topic><topic>Mice</topic><topic>Mutagenicity Tests - methods</topic><topic>Mutation</topic><topic>Phosphatidylinositol glycan complementation class A gene</topic><topic>Protein synthesis</topic><topic>Rodents</topic><topic>Sorting</topic><topic>Time Factors</topic><topic>Toxicology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kimoto, Takafumi</creatorcontrib><creatorcontrib>Suzuki, Kumiko</creatorcontrib><creatorcontrib>Kobayashi, Xiao mei</creatorcontrib><creatorcontrib>Dobrovolsky, Vasily N.</creatorcontrib><creatorcontrib>Heflich, Robert H.</creatorcontrib><creatorcontrib>Miura, Daishiro</creatorcontrib><creatorcontrib>Kasahara, Yoshinori</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Environment Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Environment Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Mutation research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kimoto, Takafumi</au><au>Suzuki, Kumiko</au><au>Kobayashi, Xiao mei</au><au>Dobrovolsky, Vasily N.</au><au>Heflich, Robert H.</au><au>Miura, Daishiro</au><au>Kasahara, Yoshinori</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl- N-nitrosourea: Direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression</atitle><jtitle>Mutation research</jtitle><addtitle>Mutat Res</addtitle><date>2011-07-14</date><risdate>2011</risdate><volume>723</volume><issue>1</issue><spage>36</spage><epage>42</epage><pages>36-42</pages><issn>1383-5718</issn><issn>0027-5107</issn><eissn>1879-3592</eissn><abstract>► We developed a flow cytometric
Pig-a assay for measuring gene mutation in the mouse. ► We used this assay to elucidate the generation of mutations in the
Pig-a assay. ► The kinetics of
Pig-a mutant manifestation is more rapid in bone marrow than in peripheral blood. ► CD24-negative erythroids have mutation in the
Pig-a gene. ► The
Pig-a assay on red blood cells is useful for the detection of
in vivo mutagenicity.
Our previous rat studies indicate that the endogenous
Pig-a gene is a promising reporter of
in vivo mutation and potentially useful as the basis for an
in vivo genotoxicity assay. The function of the Pig-a protein in the synthesis of glycosylphosphatidyl inositol (GPI) anchors is conserved in variety of eukaryotic cells, including human and rodent cells, which implies that
Pig-a mutants can be measured in a similar manner in different mammalian species. In the present study, we developed a flow cytometric
Pig-a assay for rapidly measuring gene mutation in the mouse. An antibody to TER-119, a specific cell-surface marker of murine erythroid lineage, was used to identify erythrocytes in peripheral blood (PB) and erythroids in bone marrow (BM). An antibody to CD24, a GPI-anchored protein, was used to identify
Pig-a mutants as CD24-negative cells. CD-1 mice were administered a single dose of 100
mg/kg
N-ethyl-
N-nitrosourea (ENU), and PB and BM were collected at 1, 2, and 4 weeks after dosing. While the
Pig-a mutant frequency (MF) in PB was increased moderately at 2 and 4 weeks after ENU dosing, the
Pig-a MF in BM was strongly increased starting at 1 week after the dosing, with the elevated MF persisting for at least 4 weeks after the dosing. We also used flow cytometric sorting to isolate CD24-negative erythroids from the BM of ENU-treated mice. cDNA sequencing indicated that these cells have mutations in the
Pig-a gene, with base-pair substitutions typical of ENU-induced mutation spectra. The results indicate that the
Pig-a mutation assay can be adapted for measuring mutation in BM erythroids and PB of mice. Taken together, the data suggest that
Pig-a mutants are fixed in the BM, where they further proliferate and differentiate; erythrocytes derived from these BM
Pig-a mutants transit from the BM and accumulate in PB.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>21549855</pmid><doi>10.1016/j.mrgentox.2011.03.016</doi><tpages>7</tpages></addata></record> |
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subjects | Animals Antibodies Biological and medical sciences Bone Marrow Cells - drug effects CD24 Erythrocytes Erythrocytes - drug effects Ethylnitrosourea - toxicity Flow cytometry Fundamental and applied biological sciences. Psychology Gene expression Gene mutation Genetics of eukaryotes. Biological and molecular evolution Glycosylphosphatidyl inositol Glycosylphosphatidylinositols - metabolism Medical sciences Membrane Proteins - genetics Mice Mutagenicity Tests - methods Mutation Phosphatidylinositol glycan complementation class A gene Protein synthesis Rodents Sorting Time Factors Toxicology |
title | Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl- N-nitrosourea: Direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression |
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