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 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, P(TF), was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked P(TF) 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 (P(NSE)) 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.
Leuchtenberger
S
S
Developmental Biology and Molecular Pathology, W7, University of Bielefeld, 33615 Bielefeld, Germany.
Perz
A
A
Gatz
C
C
Bartsch
J W
JW
D000818:Animals; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins; D017796:Caulimovirus / Q000235:genetics; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D004790:Enzyme Induction / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D009420:Nervous System / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology*
D001426:Bacterial Proteins; D012333:RNA, Messenger; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D013752:Tetracycline