Document Detail

Development of an in vivo gene mutation assay using the endogenous Pig-A gene: I. Flow cytometric detection of CD59-negative peripheral red blood cells and CD48-negative spleen T-cells from the rat.
MedLine Citation:
PMID:  18626999     Owner:  NLM     Status:  MEDLINE    
The product of the phosphatidylinositol glycan complementation group A gene (Pig-A) is involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors that link various protein markers to the surface of several types of mammalian cells, including hematopoietic cells. Previous observations indicate that Pig-A mutation results in the lack of GPI synthesis and the absence of GPI-anchored proteins on the cell surface. As a first step in designing a rapid assay for measuring Pig-A mutation in the rat, we developed flow cytometry (FCM) strategies for detecting GPI-negative cells in rat peripheral blood and spleen. Anti-CD59 was used to detect GPI-anchored proteins on red blood cells (RBCs), and anti-CD48 was used to detect GPI-anchored proteins on spleen T-cells. The spontaneous frequency of CD59-negative RBCs in five male F344 rats ranged from 1 x 10(-6) to 27 x 10(-6). In contrast, treatment of five rats with three doses of 40 mg/kg N-ethyl-N-nitrosourea (ENU) increased the frequency of CD59-negative RBCs to 183 x 10(-6) to 249 x 10(-6) at 2 weeks and to 329 x 10(-6) to 413 x 10(-6) at 4 weeks after dosing. In the same 4-week posttreatment rats, the frequency of CD48-negative T-cells was 11 x 10(-6) to 16 x 10(-6) in control rats and 194 x 10(-6) to 473 x 10(-6) in ENU-treated rats. The frequencies of GPI-deficient cells were similar for RBCs and spleen T-cells. These results indicate that FCM detection of GPI-linked markers may form the basis for a rapid in vivo mutation assay. Although RBCs may be useful for a minimally invasive assay, T-cells are a promising tissue for both detecting GPI-deficient cells and confirming that Pig-A gene mutation is the cause of the phenotype.
Daishiro Miura; Vasily N Dobrovolsky; Yoshinori Kasahara; Yasuhiro Katsuura; Robert H Heflich
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Environmental and molecular mutagenesis     Volume:  49     ISSN:  1098-2280     ISO Abbreviation:  Environ. Mol. Mutagen.     Publication Date:  2008 Oct 
Date Detail:
Created Date:  2008-10-13     Completed Date:  2008-10-28     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8800109     Medline TA:  Environ Mol Mutagen     Country:  United States    
Other Details:
Languages:  eng     Pagination:  614-21     Citation Subset:  IM    
Copyright Information:
Published 2008 Wiley-Liss, Inc.
Division of Genetic and Reproductive Toxicology, US Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
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MeSH Terms
Antigens, CD / immunology*
Antigens, CD59 / blood*
Erythrocytes / immunology*
Ethylnitrosourea / toxicity
Flow Cytometry / methods*
Membrane Proteins / genetics*
Mutagenicity Tests*
Mutagens / toxicity
Rats, Inbred F344
Spleen / cytology*
T-Lymphocytes / immunology*
Reg. No./Substance:
0/Antigens, CD; 0/Antigens, CD59; 0/CD48 antigen; 0/Glycosylphosphatidylinositols; 0/Membrane Proteins; 0/Mutagens; 0/phosphatidylinositol glycan-class A protein; 759-73-9/Ethylnitrosourea

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