Fig 1: Synaptotagmin 2 is ectopically overexpressed in the hippocampus, amygdala, cortical amygdala, and piriform cortex in CamiCre+ brainImmunofluorescent images of Syt2 or VGlut1 staining in brains of CamiCre mice. The white dotted boxes in the left panels are magnified in the right panels. The magenta dotted boxes are areas used for colocalization analyses in Figure 3. Amg, amygdala; Co-Amg, cortical amygdala; Hp, Hippocampus; Mo, motor cortex; Pir, piriform cortex. Scale bars: left 1 mm; right 250 µm.
Fig 2: Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of CamiCre+ cerebrum(A) Representative high-resolution immunofluorescence images from corresponding areas indicated by magenta dotted boxes in Figure 2 sp, stratum pyramidale; sl, stratum lucidum.(B) Results from colocalization analyses on a total of 6.3 × 104 µm2 per region. Ectopically expressed Syt2 showed significant colocalization with VGlut1 in CamiCre+ brain. R, Pearson correlation coefficient; M1 and M2, Manders coefficient. NS, not significant; ***p < 0.0001 (significant correlation or colocalization); †††p < 0.0001 (significant noncorrelation or noncolocalization compared to random displacement images). Scale bars: (CA1) 5 µm; (others) 10 µm.
Fig 3: BAC transgene integration sites are within the Syt2 locus on chromosome one of CamiCre+ mice(A) Results of TLA sequence coverage across the mouse genome. Chromosomes are indicated on the y axis, and the chromosomal position is shown on the x axis. The identified integration site is encircled in green. The coverage peak on chromosome 18 is owing to the homology of the BAC transgene with the endogenous genome.(B) Break points for BAC transgene integration sites were identified by TLA sequencing. Coding and noncoding regions in exons are indicated by indigo and light blue boxes, respectively. Red arrowheads indicate the three integration sites at positions 134,666,219, 134,668,306, and 134,669,891 of mouse chromosome 1 (GRC m38). See also Figure S4.
Fig 4: Synaptotagmin 2 is significantly upregulated in CamiCre+ forebrain(A) qPCR analysis of mRNA expression in the hippocampus in indicated mouse lines. Syt2-Int8 represents pre-mRNA of Syt2. Values were normalized by Gapdh expression level and are shown as fold increases against Cre- controls. Pgk1 is another housekeeping gene.(B and C) Western blotting images of different brain regions. CB, cerebellum; CC, cerebral cortex; HP, hippocampus; MB, midbrain; MO, medulla oblongata; OB, olfactory bulb.(D) Quantification results of C. Values were normalized against a-tubulin expression level and are shown as fold increases against Cre- controls.(E) Western blotting images of hippocampal lysates showing Syt2 expression levels in various Cre-expressing methodologies. Viral concentration was diluted 2x in three steps for AAV-hSyn-Cre expression. *, nonspecific band. Mean ± SD is shown. **p < 0.01; ***p < 0.001. N = 4 (biological replicates). Statistical tests were performed using two-tailed, unpaired t-tests. See also Figure S2.
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