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

Molecular Brain

Yuki Hayano¹  Yoshihisa Nakahata¹  Ryohei Yasuda¹  Hirofumi Nishizono²  Yasuhito Ishigaki² 

[1] Max Planck Florida Institute for Neuroscience;Medical Research Institute, Kanazawa Medical University;

关键词: Cre/LoxP;    floxed mouse;    CRISPR-Cas9;    genome editing;    CaMK1;   

DOI  :  10.1186/s13041-021-00859-7

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown