In vivo mutation assay based on the endogenous Pig-a locus
In vivo mutation assay based on the endogenous Pig-a locus
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DOI：
10.1002/em.20379
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发表时间：
2008-05-01
影响因子：
2.8
通讯作者：
Dertinger, Stephen D.
中科院分区：
文献类型：
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作者：
Bryce, Steven M.;Bemis, Jeffrey C.;Dertinger, Stephen D.
X染色体的Pig-a基因的产物在糖基磷脂酰肌醇（GPI）锚生物合成的第一步中起作用，并且因此对于将某些蛋白质附着到细胞表面是必需的。本文所述的实验被设计为评估缺乏GPI锚定蛋白是否可以形成体内突变测定的基础。具体地，我们使用CD 59阴性细胞表面表型来表示Pig-a突变。除抗CD 59-PE外，还使用了其他两种荧光试剂：噻唑橙子用于区分成熟红细胞、网织红细胞（RET）和白细胞;抗CD 61用于分离血小板。这些实验是用Sprague道利大鼠进行的，重点是两个细胞群，总红细胞和RET。分析方法计数CD 59阴性红细胞的能力最初通过重建实验进行评估，其中将模拟MUSCLE的细胞添加到对照血液中。随后，用模型诱变剂ENU（100 mg/kg/天）或DMBA（40 mg/kg/天）对雌性大鼠进行三次处理。在不同的时间间隔采集血液标本，最迟在暴露后6周。考虑到所有第4-6周的数据，我们发现ENU和DMBA的CD 59阴性细胞范围分别为239 - 855 x 10（-6）和82 - 405 x 10（-6）。这些值始终大于阴性对照大鼠中观察到的值（18 +/- 19 x 10（-6））。与总红细胞相比，在遗传毒性暴露动物中观察到的RET频率升高通常较高。这些数据支持的假设，一个有效的体内突变试验，可以开发围绕流式细胞计数的红细胞和/或RET表现出异常GPI锚定蛋白表达。
The product of the X-chromosome's Pig-a gene acts in the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis, and is thereby essential for attaching certain proteins to the cell surface. The experiments described herein were designed to evaluate whether lack of GPI-anchored proteins could form the basis of an in vivo mutation assay. Specifically, we used a CD59-negative cell surface phenotype to denote Pig-a mutation. Besides anti-CD59-PE, two other fluorescent reagents were used: thiazole orange to differentiate mature erythrocytes, reticulocytes (RETs), and leukocytes; and anti-CD61 to resolve platelets. These experiments were performed with Sprague Dawley rats, and focused on two cell populations, total erythrocytes and RETs. The ability of the analytical method to enumerate CD59-negative erythrocytes was initially assessed with reconstruction experiments whereby mutant-mimicking cells were added to control bloods. Subsequently, female rats were treated on three occasions with the model mutogens ENU (100 mg/kg/day) or DMBA (40 mg/kg/day). Blood specimens were harvested at various intervals, as late as 6 weeks post-exposure. Considering all week 4-6 data, we found that CD59-negative cells ranged from 239 to 855 x 10(-6) and 82 to 405 x 10(-6) for ENU and DMBA, respectively. These values were consistently greater than those observed for negative control rats (18 +/- 19 x 10(-6)). The elevated frequencies observed for the genotoxicant-exposed animals were usually higher for RETs compared to total erythrocytes. These data support the hypothesis that an efficient in vivo mutation assay can be developed around flow cytometric enumeration of erythrocytes and/or RETs that exhibit aberrant GPI-anchored protein expression.