Fig 2: Tph1 KO mice constructed using Tph1tm1Kry retains partial TPH activity. (a) Genomic structure of Tph1tm1Kry. (b) Concentration of jejunal 5-HT in wild type and Tph1tm1Kry GKO mice (n = 3–7 per group). All data are presented as means ± standard error (**P < 0.01 vs. wild type, Student’s t-test). (c) Representative immunohistochemical staining of 5-HT in the jejunum of wild type and Tph1tm1Kry GKO mice. Scale bar, 50 μm. (d) Representative immunofluorescence staining of pancreatic islets in Tph1tm1Kry βKO mice (n = 3 per group). Pancreatic sections were stained for insulin (green) and 5-HT (red). Control: MIP-Cre-hGH; βKO: Tph1tm1Kry βKO. Scale bar, 50 μm. (e) The amino acid sequence of TPH1. Red letters indicate overlapping splice sites; RD, regulatory domain; CD, catalytic domain; TD, tetramerization domain. (f) Tph1 transcripts of wild type mice and KO transcripts of Tph1tm1Kry GKO mice in the duodenum were amplified by RT-PCR using the primers, Tph1 Exon1 forward and Tph1 Exon11 reverse. Empty triangles indicate 1000 bp, and black triangles indicate 1500 bp. The full-length gel is presented in Supplementary Figure S3. (g) TPH1 (50 kD) and truncated TPH1 (37 kD) were detected by immunoblot analyses of pancreatic islets from Tph1tm1Kry βKO and hetero βKO mice. The full-length blot is presented in Supplementary Figure S2.

Fig 3: Tph1 expression and 5-HT levels in the newly constructed Tph1 floxed mouse model. (a) Tph1 mRNA levels in the duodenum, jejunum, ileum, and colon of wild type and Tph1tm1c GKO mice were determined by quantitative real time RT-PCR and are expressed relative to those of the wild type (n = 3–5 per group). (b) 5-HT levels in the duodenum, jejunum, ileum, colon, and brain were determined by LC-MS/MS (n = 3–8 per group). All data are presented as means ± standard error (**P < 0.01, ***P < 0.001 vs. wild type, Student’s t-test). (c) Representative immunohistochemical staining of 5-HT in the duodenum, jejunum, ileum, and colon of wild type and Tph1tm1c GKO mice. Scale bar, 50 μm. (d) Representative immunofluorescence staining of pancreatic islets from MIP-Cre-hGH (Control) and Tph1tm1c βKO mice (n = 3 per group). Pancreatic sections were stained for insulin (green) and 5-HT (red). Scale bar, 50 μm.

Fig 4: Construction of a new Tph1 floxed mouse model. (a) Schematic representation of the targeting strategy for generating a new Tph1 floxed mouse. (b) Genotyping of Tph1tm1a and Tph1tm1c mice. (c) Genotyping of pancreatic islets from Tph1tm1c βKO mice. Empty triangles indicate 500 bp, and black triangles indicate 1000 bp. The full-length gels are presented in Supplementary Figure S3. Representative X-gal staining of the brain (d) and small intestine (e) in wild type and Tph1tm1a mice (n = 3 per group). Red circles indicate the pineal grand. Images were captured using a stereomicroscope.