Fig 2: 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 α-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.

Fig 3: SLC6A7 protein is upregulated and shows ectopic expression in CamiCre+ hippocampus(A) Western blotting results from hippocampal lysates.(B) Quantification results of A. Values were normalized against α-tubulin expression level and are shown as fold increases against Cre- controls. Mean ± SD is shown. ∗∗p < 0.01. N = 4 (biological replicates). Statistical tests were performed using two-tailed, unpaired t-tests.(C) Immunofluorescent images of SLC6A7 or Synapsin1/2 staining in the hippocampal region of CamiCre mice. Arrows point to mossy fibers projecting from granule cells in the dentate gyrus. Scale bars: 250 μm.

Fig 4: Establishment of transgenic mice. (A) Principle of tissue-specific inducible transgenic mice system. (B) Cre was detected in lung tissue by IHC staining (scale bar, 100 µm). (C) Cre was detected by IHC staining in the peripheral lung tissue and then SPC was detected by IHC staining after antibody stripping. The solid arrows indicate that Cre and SPC were co-localized in the same cell. (D) SPC was detected by IHC staining and then mEGFP was detected by IHC staining after antibody stripping. The solid arrows indicate that SPC and mEGFP were co-localized in the same cell, while the hollow arrows indicate that SPC and mEGFP were not co-localized in the same cell (scale bars, 50 µm). IHC, immunohistochemistry; mEGFP, cell membrane-localized enhanced green fluorescence protein; SPC, surfactant protein C; ACTB, β-actin.

Fig 5: CamiCre+ mice show hyperactivity and enhanced learning in fear conditioning(A–D) Open field test.(E–H) Cued and contextual fear conditioning. Mean ± SEM is shown. NS, not significant; ∗p < 0.05; ∗∗p < 0.01. p values labeled in the line graphs indicate the genotype effect of two-way ANOVA; (F) F (1, 37) = 5.023, (H) F (1, 37) = 6.204. N = 18 for Cre−, N = 24 for Cre+ for open field test and N = 17 for Cre−, N = 22 for Cre+ for cued and contextual fear conditioning. Statistical tests were performed using two-tailed, unpaired t-tests (A-E), two-tailed Welch’s t tests (G), or a two-way ANOVA (F, H). Mice that showed significant freezing before the first conditioned stimulus were excluded from the fear conditioning analysis. See also Figure S5.