Shimizu Y, Shimizu N
Biochem Genet. 1981 Feb;19(1-2):95-106. doi: 10.1007/BF00486140.
An immunocytochemical method has been devised which allows the screening of a large number of human x mouse cell hybrid colonies for the retention of a specific human chromosomal gene and the presence of its translation product. The glycolytic enzyme phosphoglucose isomerase (PGI; D-glucose-6-phosphate ketol-isomerase; EC 5.3.1.9) was chosen as a marker which is known to be controlled by the gene on human chromosome 19. The technique involves three steps: (i) immobilization of growing cell colonies in agar gel containing antibody that specifically reacts with human-type PGI; (ii) lysis of the embedded cells with Triton X-100 to release enzyme antigens and precipitate as an immune complex; and (iii) visualization of the antibody-fixed enzymes by histochemical activity staining. Human PGI activity released from a colony consisting of as few as eight cells generated an adequate signal. Variation of intensity was noticed and attributed to gene dosage in individual cells. The percentage of human PGI-positive colonies in each of nine independent hybrid lines estimated by this method generally paralleled the frequency of retention of human chromosome 19 determined by conventional karyotyping. The technique can be applied to many other markers and be used as "a half-selection" system in combination with the "replica plating" method.
已设计出一种免疫细胞化学方法，可用于筛选大量人 - 小鼠细胞杂交菌落，以检测特定人类染色体基因的保留情况及其翻译产物的存在。糖酵解酶磷酸葡萄糖异构酶（PGI；D - 葡萄糖 - 6 - 磷酸酮醇异构酶；EC 5.3.1.9）被选作标记物，已知该酶受人类19号染色体上的基因控制。该技术包括三个步骤：（i）将生长中的细胞菌落固定在含有与人型PGI特异性反应的抗体的琼脂凝胶中；（ii）用Triton X - 100裂解包埋的细胞，释放酶抗原并沉淀形成免疫复合物；（iii）通过组织化学活性染色使抗体固定的酶可视化。由少至八个细胞组成的菌落释放出的人PGI活性产生了足够的信号。注意到强度存在差异，并归因于单个细胞中的基因剂量。通过该方法估计的九个独立杂交系中每个人PGI阳性菌落的百分比通常与通过传统核型分析确定的人类19号染色体保留频率平行。该技术可应用于许多其他标记物，并可与“复制平板”方法结合用作“半选择”系统。
