Killary A M, Lugo T G, Fournier R E
Biochem Genet. 1984 Apr;22(3-4):201-13. doi: 10.1007/BF00484224.
The photosensitivity of bromodeoxyuridine (BrdU)-substituted cells is known to be markedly enhanced by the fluorochrome Hoechst 33258. Since the incorporation of BrdU into nucleic acids depends upon its prior phosphorylation via thymidine kinase (TK; EC 2.7.1.21), cells deficient in TK activity are refractory to photoinduced killing. These observations suggested that combined treatment with BrdU, Hoechst 33258, and visible light would constitute an efficient selective strategy for the recovery of TK- mutant cells. In this report we describe a single-step selection protocol which reduced the survival of TK+ cells by a factor of 10(5) without affecting the viability of TK- mutants. This procedure was used to isolate H4IIEC3 -derived rat hepatoma cells deficient in TK activity. The properties of several TK- hepatoma clones are discussed.
已知荧光染料Hoechst 33258可显著增强溴脱氧尿苷（BrdU）取代细胞的光敏性。由于BrdU掺入核酸取决于其先通过胸苷激酶（TK；EC 2.7.1.21）进行磷酸化，缺乏TK活性的细胞对光诱导杀伤具有抗性。这些观察结果表明，BrdU、Hoechst 33258和可见光联合处理将构成一种恢复TK突变细胞的有效选择策略。在本报告中，我们描述了一种单步选择方案，该方案可使TK+细胞的存活率降低10^5倍，而不影响TK-突变体的活力。此程序用于分离缺乏TK活性的源自H4IIEC3的大鼠肝癌细胞。讨论了几个TK-肝癌克隆的特性。
