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Optimized self-excising Cre-expression cassette for mammalian cells
Anssi J Mähönen1, Kari J Airenne, Miia M Lind
1Department of Molecular Medicine and Biotechnology, AI Virtanen Institute, University of Kuopio, Kuopio, Finland.
Biochemical and Biophysical Research Communications
|June 29, 2004
Summary
Researchers developed a novel silent self-inactivating Cre (SSi-Cre) expression cassette to overcome Cre recombinase toxicity in mammalian cells and leaky expression in bacteria, improving Cre/loxP construct cloning.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Cre recombinase is crucial for site-specific DNA recombination in the Cre/loxP system.
- Overexpression of Cre recombinase in mammalian cells causes toxicity.
- Leaky Cre expression in Escherichia coli complicates the cloning of Cre/loxP constructs.
Purpose of the Study:
- To develop a novel expression cassette to address Cre recombinase toxicity and leaky expression.
- To facilitate the cloning and application of Cre/loxP systems.
Main Methods:
- Construction of a silent self-inactivating Cre (SSi-Cre) expression cassette.
- Modification of loxP sites flanking a Cre/Int/DsRed fusion gene.
- Inclusion of an intron within the Cre coding sequence to prevent bacterial expression.
- Incorporation of a reporter gene for visualizing Cre activity.
Main Results:
- The SSi-Cre cassette effectively prevents leaky Cre expression in E. coli.
- The system resolves toxicity issues associated with Cre overexpression in mammalian cells.
- A DsRed reporter gene allows for visualization of Cre activity via fluorescent microscopy.
Conclusions:
- The SSi-Cre cassette offers a universal and improved strategy for generating and utilizing Cre/loxP constructs.
- This system provides a solution for Cre toxicity and expression issues, enhancing its utility in various research applications.