Generation of Flag/DYKDDDDK Epitope Tag Knock-In Mice Using i -GONAD Enables Detection of Endogenous CaMKIIα and β Proteins.

Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIβ mice by combining CRISPR-Cas9 genome editing with electroporation ( i -GONAD). With i -GONAD, it is possible to insert a small fragment of up to 200 bp into the genome of the target gene, enabling efficient and convenient tagging of a small epitope. Experiments with commercially available anti-Flag antibodies could readily detect endogenous CaMKIIα and β proteins by Western blotting, immunoprecipitation, and immunohistochemistry. Our data demonstrated that the generation of Flag/DYKDDDDK tag knock-in mice by i -GONAD is a useful and convenient choice, especially if specific antibodies are unavailable.