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Journal Article

Rapid generation of conditional knockout mice using the CRISPR-Cas9 system and electroporation for neuroscience research

MPS-Authors

Nishizono,  Hirofumi
Max Planck Florida Institute for Neuroscience, Max Planck Society;

Hayano,  Yuki
Max Planck Florida Institute for Neuroscience, Max Planck Society;

Nakahata,  Yoshihisa
Max Planck Florida Institute for Neuroscience, Max Planck Society;

Ishigaki,  Yasuhito
Max Planck Florida Institute for Neuroscience, Max Planck Society;

Yasuda,  Ryohei
Max Planck Florida Institute for Neuroscience, Max Planck Society;

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Citation

Nishizono, H., Hayano, Y., Nakahata, Y., Ishigaki, Y., & Yasuda, R. (2021). Rapid generation of conditional knockout mice using the CRISPR-Cas9 system and electroporation for neuroscience research. Molecular Brain, (1). Retrieved from https://molecularbrain.biomedcentral.com/track/pdf/10.1186/s13041-021-00859-7.


Cite as: https://hdl.handle.net/21.11116/0000-000C-DFB0-2
Abstract
The Cre/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of CaMK1 in a short period of 125 days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL/6 N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.