O'Neill J P, McGinniss M J, Berman J K, Sullivan L M, Nicklas J A, Albertini R J
University of Vermont, Genetics Laboratory, Burlington 05401.
Mutagenesis. 1987 Mar;2(2):87-94. doi: 10.1093/mutage/2.2.87.
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 (TG1) phenotype, lack hypoxanthine guanine phosphoribosyl transferase (HPRT) activity and thus appear to be the result of in vivo somatic cell mutation events. In order to employ this T-lymphocyte cloning assay for quantitative determination of the in vivo TGr mutant 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 TGr mutant cell cloning at densities greater than 2 x 10(4) cells 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 the in vivo TGr mutant frequency in human T-lymphocytes.
通过在96孔微量滴定板中进行有限稀释来克隆细胞，已被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。这些集落显示出对TG抗性（TG1）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淋巴细胞克隆试验来定量测定人体内TG抗性（TGr）突变频率，我们确定了非选择细胞和TG选择细胞形成T细胞集落的最佳条件。所研究的参数包括培养基、血清、促有丝分裂原植物血凝素的量、T细胞生长因子（TCGF）的量以及经辐照的饲养细胞或辅助细胞的数量。在最佳条件下，无论是非选择条件还是TG选择条件，阳性孔的比例都与每孔接种的细胞数成正比。因此，T细胞克隆效率与接种物大小无关。有证据表明，当密度大于每圆底孔2×10⁴个细胞时，TGr突变细胞克隆会下降，这可能是由于野生型细胞和突变细胞之间的代谢合作所致。本研究中确定的条件似乎为定量测定人T淋巴细胞中的体内TGr突变频率提供了一种方法。
