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. 2019 Nov 14;9:16843. doi: 10.1038/s41598-019-53317-9

Figure 1.

Figure 1

Generation of Caskin dKO mice. (A–D) Gene targeting strategies to knockout exons 2 to 6 of Caskin1 (A,B) or exons 3 to 7 of Caskin2 (C,D). In the targeting vectors, exons to be deleted were flanked by loxP sites. To achieve positive and negative selection, a neomycin (Neo) resistance gene cassette was inserted into intron 1 and the thymidine kinase gene (TK) was inserted downstream of exon 7 (Caskin1) or 8 (Caskin2), respectively. Mice carrying mutant floxed allele were crossed with transgenic C57Bl/6 (Bl6) mouse carrying Cre recombinase. (B) Position of deleted exons 2 to 6 are represented in chromosome 17. The primer set (a1, s1, s2) and the amplified regions (WT: 306 bp, KO: 366 bp) are marked on the wild type (WT) and KO locus. Right panel shows PCR genotyping of wild type (+/+), homozygous Caskin1 KO (−/−) and heterozygous (+/−) mice. (D) Position of deleted exons 3 to 7 are represented in chromosome 11. The primer set (a2, s3, s4) and the amplified regions (WT: 255 bp, KO: 315 bp) are marked on the wild type (WT) and KO sites. Right panel shows PCR genotyping of wild type (+/+), homozygous Caskin1 KO (−/−) and heterozygous (+/−) mice. (E-F) Comparison of endogenous Caskin1 levels between C57Bl6/J wild type (WT), heterozygous (dHZ) and Caskin dKO (dKO) mice at the age of 3 months. Quantitative data was based on 3 independent samples by normalizing Caskin1/βIII-tubulin ratio. (G) Median-sagittal sections of WT and dKO brains obtained at 3-months of age and stained by gallocyanine-chrome alum stains. Scale bar: 3 mm. (H–I) Total ambulation distance (H) and velocity (I) within the open field during aging. Caskin dHZ (n = 16; white columns) and Caskin dKO (n = 16; grey columns) data are displayed. All data are shown as mean ± SEM. *p < 0.05; ***p < 0.001.