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Free keywords:
Animals, Mice, Mice, Inbred C57BL, Integrases, Gene Targeting, Mice, Knockout, Electroporation, Neurosciences, CRISPR-Cas Systems, Transgenes, Exons, Base Sequence, Calcium-Calmodulin-Dependent Protein Kinase Type 1, CaMK1, Cre/LoxP, CRISPR-Cas9, Embryo Transfer, floxed mouse, Gene Editing, genome editing
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.
