Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells

Conditional expression of suicide genes in vivo has a wide range of applica tions in biological research and requires a minimal basal promoter activity In the uninduced state. To reduce basal activity of tetracycline (tc)-indu cible target promoters we combined synthetic tet operators in varying numbe rs with a core promoter derived from the plant viral 35S promoter. An optim ized promoter, P-TF, was found to exert a stringent regulation of luciferas e in combination with tTA and rtTA in different mammalian cell lines. We li nked P-TF to the barnase gene, coding for a highly active RNase from Bacill us amyloliquefaciens. Stable cell clones expressing barnase under control o f tTA exerted cell death only after tc withdrawal, correlating with a 10-fo ld induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (P-NSE) leads to barnase expression and c ell death in neuronal cells after tc withdrawal. Taken together, our data d emonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene prop agation in both cell lines and in transgenic mice are observed.