Isolation of thymidine kinase-deficient rat hepatoma cells by selection with bromodeoxyuridine, Hoechst 33258, and visible light.
Abstact
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.