Optimized self-excising Cre-expression cassette for mammalian cells.
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.
Mähönen
Anssi J
AJ
Department of Molecular Medicine and Biotechnology, AI Virtanen Institute, University of Kuopio, Kuopio, Finland.
Airenne
Kari J
KJ
Lind
Miia M
MM
Lesch
Hanna P
HP
Ylä-Herttuala
Seppo
S
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D019426:Integrases / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D017931:DNA Primers; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases