Nucleic Acids Research, 2001, Vol. 29, No. 16 e76
© 2001 Oxford University Press
Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells
Developmental Biology and Molecular Pathology, W7, University of Bielefeld, 33615 Bielefeld, Germany and 1Albrecht-Haller Institute for Plant Sciences, University of Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany
Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state. To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter. An optimized promoter, PTF, was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked PTF to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens. Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (PNSE) leads to barnase expression and cell death in neuronal cells after tc withdrawal. Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed.
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