Revollo Javier, Pearce Mason G, Petibone Dayton M, Mittelstaedt Roberta A, Dobrovolsky Vasily N
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA.
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA
Mutagenesis. 2015 May;30(3):315-24. doi: 10.1093/mutage/geu030. Epub 2015 Mar 27.
The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene.
Pig-a检测用于监测实验动物和人类的体细胞突变。该检测通过流式细胞术检测糖基磷脂酰肌醇（GPI）锚定蛋白表面标志物缺陷的造血细胞。然而，鉴于蛋白标志物的合成（及其基因的表达）独立于X连锁的Pig-a基因的表达及其酶产物的功能，细胞表面标志物的缺陷可能由多种事件引起（例如，由标志物基因本身或参与GPI合成的大约二十几个常染色体基因中的任何一个的突变或表观遗传沉默引起）。在这里，我们提供了直接证据，表明大鼠T细胞中GPI锚定表面标志物CD48的缺陷与内源性X连锁Pig-a基因的突变有关。我们用N-乙基-N-亚硝基脲（ENU）处理雄性F344大鼠，并从流式细胞术鉴定和分选的CD48缺陷脾T淋巴细胞建立菌落。分子分析证实，扩增的分选细胞在Pig-a基因中有突变。我们模型中Pig-a突变的谱与在其他体内模型中确定的ENU诱导突变的谱一致，主要是A:T处的碱基对替换，Pig-a基因组DNA的非转录链上的突变T。我们还使用下一代测序从由流式分选的CD48缺陷淋巴细胞产生的64个克隆库中得出类似的突变谱。我们的发现证实，Pig-a检测能够检测到其设计要检测的东西——Pig-a基因中的基因突变。
