Analysis of mutation in the rat Pig‐a assay: II. Studies with bone marrow granulocytes

Abstract

The in vivo erythrocyte Pig-a gene mutation assay measures the phenotypic loss of GPI-anchored surface markers. Molecular analysis of the marker-deficient erythrocytes cannot provide direct proof that the mutant phenotype is due to mutation in the Pig-a gene because mammalian erythrocytes lack genomic DNA. Granulocytes are nucleated cells that originate from myeloid progenitor cells in bone marrow as is the case for erythrocytes, and thus analysis of Pig-a mutation in bone marrow granulocytes can provide information about the source of mutations detected in the erythrocyte Pig-a assay. We developed a flow cytometric Pig-a assay for bone marrow granulocytes and evaluated granulocyte Pig-a mutant frequencies in bone marrow from male rats treated acutely with N-ethyl-N-nitrosourea (ENU). Bone marrow cells from these rats were stained with anti-CD11b for identifying granulocytes and anti-CD48 for detecting the Pig-a mutant phenotype. The average Pig-a mutant frequency in granulocyte precursors of control rats was 8.42 × 10 , whereas in ENU-treated rats it was 567.13 × 10 . CD11b-positive/CD48-deficient mutant cells were enriched using magnetic separation and sorted into small pools for sequencing. While there were no Pig-a mutations found in sorted CD48-positive wild-type cells, Pig-a mutations were detected in mutant granulocyte precursors. The most frequent mutation observed was T→A transversion, followed by T→C transition and T→G transversion, with the mutated T on the nontranscribed DNA strand. While the spectrum of mutations in bone marrow granulocytes was similar to that of erythroid cells, different Pig-a mutations were found in mutant-phenotype granulocytes and erythroids from the same bone marrow samples, suggesting that most Pig-a mutations were induced in bone marrow cells after commitment to either the granulocyte or erythroid developmental pathway. Environ. Mol. Mutagen. 59:733-741, 2018. © 2018 Wiley Periodicals, Inc.

Analysis of mutation in the rat Pig‐a assay: I) studies with bone marrow erythroid cells

“…). The spectrum of ENU‐induced Pig‐a mutations in bone marrow granulocytes from the same rats was remarkably similar, although the mutations occurred not necessarily at the same positions within the Pig‐a gene [for details see the companion manuscript by Dad et al, ]. Such a spectrum is consistent with the mutagenic properties of ENU determined in other in vivo models that detect mutation in the X‐linked Hprt and Pig‐a reporter genes by conventional Sanger‐sequencing of expanded mutant clones [Jansen et al, ; Casciano et al, ; Miura et al, ; Revollo et al, ].…”

“…As an example, anti‐rat erythroid OX‐83 antibody may have altered the performance of the anti‐CD59 antibody in the rat BME Pig‐a assay, but Kimoto et al, described a mouse BME Pig‐a assay in which anti‐mouse erythroid Ter‐119 antibody was successfully used for positive identification of erythroid nucleated cells [Kimoto et al, ]. Another example of antibody interference was noted in our laboratory during the development of a rat bone marrow granulocyte Pig‐a assay [Dad et al, ], when inclusion of an antibody (produced by clone HIS48) for labeling a secondary lineage‐specific granulocyte marker interfered with the performance of an antibody against the GPI‐anchored CD48 marker (prevented the detection of potential CD48‐deficient mutants; see the accompanying manuscript on bone marrow granulocyte Pig‐a assay). Unless, of course, the epitope for HIS48 antibody happens to be GPI‐anchored.…”

“…Additional studies with mutagens other than ENU, and with myeloid lineage cells related to BMEs, such as bone marrow granulocytes, will further reinforce the linkage between mutation in the Pig‐a gene and the phenotype measured in the erythrocyte Pig‐a assay. A study involving bone marrow granulocyte analyses was conducted in parallel with this BME study and is presented in an accompanying paper by Dad et al [].…”

“…The pellet was resuspended in exactly 4.5 ml of DPBS/2%FBS and stored on ice or at +4°C until ready for antibody labeling (for up to 20 hr). Some of the flushed bone marrow samples were split for use in bone marrow granulocyte analyses described in the companion paper by Dad et al [].…”

CD59‐deficient bone marrow erythroid cells from rats treated with procarbazine and propyl‐nitrosourea have mutations in thePig‐agene

“…Analyses of mutations were performed using the previously described protocol, Mutation Analysis with Multiplexed Libraries, MAML (Revollo et al ., 2017a; 2018; 2019; Dad et al ., 2018). Briefly, from 300 to 1,500 cells were sorted in bulk into a tube filled with 50 μl of DNAzol® Direct (Molecular Research Center, Cincinnati, OH).…”