Further Development of the Rat Pig-a Mutation Assay: Measuring Rat Pig-a Mutant Bone Marrow Erythroids and a High Throughput Assay for Mutant Peripheral Blood Reticulocytes
Further Development of the Rat Pig-a Mutation Assay: Measuring Rat Pig-a Mutant Bone Marrow Erythroids and a High Throughput Assay for Mutant Peripheral Blood Reticulocytes
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DOI：
10.1002/em.20677
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发表时间：
2011-12-01
影响因子：
2.8
通讯作者：
Kasahara, Yoshinori
中科院分区：
文献类型：
--
作者：
Kimoto, Takafumi;Chikura, Satsuki;Kasahara, Yoshinori
最近的研究表明，猪-a试验是一个很有前途的工具，以评估体内突变性。我们开发了一种新的大鼠Pig-a检测方法，可以方便地测量两种早期出现的血细胞群体，骨髓红细胞（BMEs）和外周血网状细胞（ret）的突变频率。在这些实验中，使用一种针对大鼠红细胞特异性标记物HIS49的抗体来鉴定红系骨髓细胞和PB红细胞（rbc）。此外，通过对转铁蛋白受体CD71（一种表达于BMEs和ret表面，而不表达于成熟红细胞表面的转铁蛋白受体）阳性细胞的磁分离，PB选择性富集了ret。通过磁富集，流式细胞术可以在5 - 8分钟内评估超过1 × 10(6)个cd71阳性ret的突变频率。40 mg/kg n -乙基-n -亚硝基脲给药1周后，检测到cd59缺陷RET和BME频率分别大于100 × 10（-6）和80 × 10(-6)；相比之下，这些大鼠中cd59缺陷总红细胞的频率为13.2 x 10（-6）。自发性猪a突变ret和BMEs的频率分别小于5 × 10（-6）和15 × 10（-6）。由于BME部分中98%的有核细胞是红母细胞，因此应该可以使用BME来确定红系细胞中cd59缺陷的猪-a突变的谱。同时对RETs和BMEs进行猪-a试验可能有助于评估化学物质的体内诱变性，特别是当长期突变表现不可行或需要确认突变诱导时。环绕。生物工程学报，2011,32(2):774-783。出版于2011年Wiley期刊公司。
Recent studies indicate that the Pig-a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig-a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid-specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 x 10(6) CD71-positive RETs could be evaluated by flow cytometry for Pig-a mutant frequency within 5 to 8 min. CD59-deficient RET and BME frequencies of more than 100 x 10(-6) and 80 x 10(-6) were detected 1 week after treating rats with 40 mg/kg N-ethyl-N-nitrosourea; by comparison, the frequency of CD59-deficient total RBCs in these rats was 13.2 x 10(-6). The frequency of spontaneous Pig-a mutant RETs and BMEs was less than 5 x 10(-6) and 15 x 10(-6), respectively. Since similar to 98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59-deficient Pig-a mutations in cells of erythroid lineage. Conducting concurrent Pig-a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary. Environ. Mol. Mutagen. 52:774-783, 2011. Published 2011 Wiley Periodicals, Inc.