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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Bloom's syndrome gene-deficient phenotype in mouse primary cells induced by a modified tetracycline-controlled trans-silencer.

We recently reported genome-wide bi-allelic mutagenesis and phenotype-based genetic screening by tetracycline-regulated disruption of the Bloom's syndrome gene (Blm) in mouse embryonic stem (ES) cells. However, the same approach was hampered in mouse tissues owing to leaky expression of the Blm gene, which is the major obstacle in the tetracycline regulatory system. Here we describe a single-chain reverse tetracycline-controlled trans-silencer (sc rtTS) which reduces leaky expression in the tet-off system. The sc rtTS consists of two silencer moieties linked by a 36 amino acid linker. Although the silencer moiety contained a dimerization domain compatible with the tetracycline-controlled transactivator (tTA), heterodimerization with tTA was prevented because intramolecular self-assembly between linked silencer moieties was preferred. The system was applied to mouse splenic lymphocytes and elevation of sister chromatid exchange, the hallmark of Blm dysfunction, was observed in the presence of doxycycline. A cassette containing both sc rtTS and tTA was introduced into the Blm allele in ES cells and reduction of basal activity was observed upon doxycycline treatment. Our data demonstrate effectiveness of sc rtTS in the tet-off system. Application of sc rtTS in mice may allow us to implement bi-allelic mutagenesis in vivo.[1]


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