Biochemical Genetics

, Volume 22, Issue 3, pp 201–213

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

Authors

  • A. M. Killary
    • Department of Microbiology and the Comprehensive Cancer CenterUniversity of Southern California School of Medicine
  • T. G. Lugo
    • Department of Microbiology and the Comprehensive Cancer CenterUniversity of Southern California School of Medicine
  • R. E. K. Fournier
    • Department of Microbiology and the Comprehensive Cancer CenterUniversity of Southern California School of Medicine
Article

DOI: 10.1007/BF00484224

Cite this article as:
Killary, A.M., Lugo, T.G. & Fournier, R.E.K. Biochem Genet (1984) 22: 201. doi:10.1007/BF00484224

Abstract

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 105 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.

Key words

somatic cell mutantsthymidine kinaserat hepatomatransfection
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Copyright information

© Plenum Publishing Corporation 1984