Dobrovolsky Vasily N, Cao Xuefei, Bhalli Javed A, Heflich Robert H
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR, USA.
Toxicology/Safety Assessment, Covance Laboratories Inc., Greenfield, IN, USA.
Methods Mol Biol. 2020;2102:315-331. doi: 10.1007/978-1-0716-0223-2_18.
The endogenous X-linked phosphatidyl inositol glycan class A gene (Pig-a) can be used as a reporter of in vivo somatic cell mutation in rats and mice. Pig-a mutant cells are deficient in specific protein surface markers and can be identified and quantified by immunofluorescent staining followed by high-throughput flow cytometry. Pig-a mutation detection is commonly performed with red blood cells (RBCs) because: (1) the low volumes of blood required for determining mutant frequencies in RBCs allow multiple samplings on small laboratory animals over extended periods of time; (2) the execution of the RBC assay is easy and the interpretation of the results is straightforward; and (3) RBC Pig-a mutant frequencies are known within hours of sample collection. Two endpoints are determined in the assay: the frequency of mutant total RBCs and the frequency of mutant reticulocytes. When Pig-a mutation is used to assess the in vivo mutagenic potential of suspect hazards, the frequency of mutant reticulocytes is an early indicator of mutagenic potential, while the mutant frequency in total RBCs can be measured more rapidly and with greater precision.
内源性X连锁磷脂酰肌醇聚糖A类基因（Pig-a）可作为大鼠和小鼠体内体细胞突变的报告基因。Pig-a突变细胞缺乏特定的蛋白质表面标志物，可通过免疫荧光染色随后进行高通量流式细胞术来识别和定量。Pig-a突变检测通常使用红细胞（RBC）进行，原因如下：（1）测定红细胞中突变频率所需的血量少，这使得能够在较长时间内对小型实验动物进行多次采样；（2）红细胞检测操作简便，结果解释直接明了；（3）在采集样本后数小时内即可得知红细胞Pig-a突变频率。该检测确定两个终点：突变总红细胞的频率和突变网织红细胞的频率。当使用Pig-a突变来评估可疑危害物的体内致突变潜力时，突变网织红细胞的频率是致突变潜力的早期指标，而总红细胞中的突变频率可以更快速且更精确地测量。
