Refinement of a T-lymphocyte cloning assay to quantify the in vivo thioguanine-resistant mutant frequency in humans.
Refinement of a T-lymphocyte cloning assay to quantify the in vivo thioguanine-resistant mutant frequency in humans.
复制标题
改进 T 淋巴细胞克隆测定，以量化人类体内硫鸟嘌呤抗性突变频率。
DOI：
10.1093/mutage/2.2.87
复制
发表时间：
1987
期刊：
影响因子：
2.7
通讯作者：
Albertini,RJ
中科院分区：
文献类型：
--
作者：
O'Neill,JP;McGinniss,MJ;Berman,JK;Sullivan,LM;Nicklas,JA;Albertini,RJ
通过在 96 孔微量滴定板中进行有限稀释进行细胞克隆已用于分离对嘌呤类似物 6-硫鸟嘌呤 (TG) 具有抗性的人 T 淋巴细胞集落。这些菌落显示出TG抗性（TGr）表型的稳定性，缺乏次黄嘌呤鸟嘌呤磷酸核糖转移酶（HPRT）活性，因此似乎是体内体细胞突变事件的结果。为了利用这种 T 淋巴细胞克隆测定来定量测定人类体内 TG 突变频率，我们定义了非选择细胞和 TG 选择细胞形成 T 细胞集落的最佳条件。研究的参数包括培养基、血清、促细胞分裂素植物血凝素的量、T细胞生长因子（TCGF）的量以及受辐射的饲养细胞或辅助细胞的数量。在最佳条件下，在非选择和 TG 选择条件下，阳性孔的比例与每孔铺板的细胞数成正比。因此，T 细胞克隆效率与接种量无关。有一些证据表明，TGr 突变细胞克隆在每圆底孔 >2 × 104 个细胞的密度下会下降，这可能是由于野生型和突变细胞之间的代谢合作所致。本研究中定义的条件似乎提供了人T淋巴细胞体内TG突变频率的定量测量。
Cell cloning by limiting dilution in 96-well microtiter plates has been employed to isolate colonies of human T-lymphocytes resistant to the purine analogue, 6-thioguanine (TG). These colonies show stability of the TG-resistant (TGr) phenotype, lack hypoxanthine guanine phosphoribosyl transferase (HPRT) activity and thus appear to be the result ofin vivosomatic cell mutation events. In order to employ this T-lymphocyte cloning assay for quantitative determination of thein vivoTGrmutant frequency in humans, we have defined the optimal conditions for T-cell colony formation with both nonselected and TG-selected cells. The parameters investigated include medium, serum, amount of the mitogen phytohaemagglutinin, amount of T-cell growth factor (TCGF) and the number of irradiated feeder or accessory cells. Under the optimal conditions, the fraction of positive wells is proportional to the number of cells plated per well with both nonselection and TG selection conditions. T-cell cloning efficiencies therefore are independent of inoculum size. There was some evidence for a decline in TGrmutant cell cloning at densities >2 × 104cells per round-bottom well, possibly due to metabolic cooperation between wild-type and mutant cells. The conditions defined in this study appear to provide a quantitative measurement of thein vivoTGrmutant frequency in human T-lymphocytes.