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

Abstract

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.

Keywords: AlphaFold2; CRISPR-Cas9 genome editing; Western blot; flag epitope tag; i-GONAD; immunohistochemistry.

Figure 1

Flag-CaMKIIα and-β protein structures predicted by AlphaFold2. (a) Flag-tagged CaMKIIα and CaMKIIβ protein structures with a Flag tag (red), kinase domain (brown), regulatory segment (green), linker region (LR, blue), and association domain (pink). (b) 3D structure of CaMKIIα (left) and Flag-CaMKIIα (right) proteins by ColabFold. (c). The 3D structure of CaMKIIβ (left) and Flag-CaMKIIβ (right) proteins using ColabFold. The prediction quality as per residue confidence score, pLDDIT, is shown as colors of 3D structure, very high (blue, pLDDT > 90), confidence (light blue, 90 > Plddt > 70), low (yellow, 70 > pLDDT > 50), and very low (red, pLDDT > 50).

Figure 2

Generation of Flag knock-in mice for CaMKIIα and CaMKIIβ. (a) Sequence of exon 1 of mouse CaMKIIα, gRNA target site (black underline), and ssODN. The square box shows protospacer adjacent motif (PAM) sequence. The start codon encoding methionin (Met) is highlighted with green. (b) Confirmation of knock-in sequences. PCR products using genomic DNA as a template were cloned and sequenced. (c) Gel electrophoresis of PCR products amplified with primer sets of CaMKIIα-Ex1-105F/R in (a). Black and blue colored IDs indicate wild-type (WT) and knock-in (KI) pups, respectively. Black, red, and blue arrows show wild-type (105 bp), KI (129 bp) and heteroduplexed band of wild-type and KI DNA fragments, respectively. (d–f) Sequence of exon 1 of mouse CaMKIIβ, gRNA target site (black underline), and ssODN. Square box shows PAM sequence. The start codon encoding methionin (Met) is highlighted with green. (e) Confirmation of knock-in sequences. (f) Gel electrophoresis of PCR products amplified with primer sets of CaMKIIβ-Ex1-100F/R in (d). Black- and blue-colored IDs indicate wild-type (WT) and knock-in (KI) pups, respectively. Black, red, and blue arrows show WT (100 bp), KI (124 bp) and heteroduplexed band, respectively. (g) A summary table of generating flag knock-in mice by i-GONAD.

Figure 3

Expression of Flag-CaMKIIα and β and association between Flag-CaMKIIα and endogenous CaMKIIβ. (a) Immunoblotting of cortical, hippocampal, and cerebellar samples in CaMKIIα^N-Flag/+ heterozygous and CaMKIIβ^{N-Flag/N-Flag} homozygous knock-in mice using anti-Flag antibody. GAPDH was used as internal control. (b) Immunoblotting using anti-Flag, CaMKIIα, and CaMKIIβ specific antibodies, after immunoprecipitation of cortex sample in CaMKIIα mouse using flag antibody (Flag IP). WCL, whole cell lysate. Asterisk shows shifted band of Flag-CaMKIIα protein, compared to that of endogenous CaMKIIα protein.

Figure 4

Detection of Flag-CaMKIIα and Flag-CaMKIIβ by immunohistochemistry. Immunostaining of hippocampal CA1 region (a–c), cortex (d–f), and cerebellum (g–i) of wild-type (WT, (a,d,g)), CaMKIIα^N-Flag (b,e,h), and CaMKIIβ^N-Flag (c,f,i) using anti-Flag (green) and neural marker anti-NeuN (red) antibodies. DAPI (blue) is nuclear marker. SO, stratum oriens, SP, stratum pyramidal, SR, stratum radiatum, SLM, stratum lacunosum-moleculare. Cortical layer I, II, III, and IV in D. ML, molecular layer, PCL, Purkinje cell layer, and GCL, granule cell layer. (j–o) Staining of anti-CaMKIIα and anti-Flag/DYKDDDDK antibodies showed complete overlap in cytoplasmic region of hippocampal CA1 (j,l,n) and dentate gyrus (DG) (k,m,o) of CaMKIIα^N-Flag/+ mice. Scale bar = 100 μm in (a–i), 50 μm in (j–o).

Figure 5

Thei-GONAD efficiency for knock-out (KO), knock-in, and conditional KO. Sematic representation of genome editing efficiency and the size of knock-in fragment.