Optimized self-excising Cre-expression cassette for mammalian cells |
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Authors: | Mähönen Anssi J Airenne Kari J Lind Miia M Lesch Hanna P Ylä-Herttuala Seppo |
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Affiliation: | Department of Molecular Medicine and Biotechnology, AI Virtanen Institute, University of Kuopio, Kuopio, Finland. |
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Abstract: | We observed that overexpression of Cre recombinase in 293 T cells has toxic effects and that the chicken beta-actin promoter is active in Escherichia coli, causing expression of Cre in bacteria. This led to significant problems in the cloning of Cre/loxP constructs. Leaky Cre-expression in E. coli, and toxicity of the Cre overexpression in mammalian cells, were solved by constructing a novel silent self-inactivating Cre (SSi-Cre) expression cassette. The SSi-Cre is based on modified loxP sites flanking the Cre/Int/DsRed fusion gene containing a Cre coding sequence interrupted by an intron, which prevents leaky expression of Cre in E. coli. Additionally, this system contains a reporter gene to visualize Cre activity by fluorescent microscopy. The SSi-Cre cassette provides a universal strategy for the generation of Cre/loxP constructs, as well as a solution to the toxicity caused by the overexpression of Cre in target cells. SSi-Cre should thus provide a useful tool for various applications based on the Cre/loxP system. |
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Keywords: | Cre loxP Recombination CAGGS Toxic Intron |
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