Fig. 1. Generation and characterization of transgenic mice for the simultaneous ablation of islet α-cells, δ-cells and γ-cells.
a, CRISPR–Cas9 strategy to replace the Sst coding sequence (Sst CDS) on mouse chromosome 16 with the human DTR coding region (targeting vector). LHA, left homology arm; RHA, right homology arm; UTR, untranslated region. b, Transgenes required for DTR-mediated non-β-cell ablation. c, PCR products of Gcg-DTR (~800 bp), Sst-DTR (WT 871 bp, KI 483 bp) and Ppy-DTR (WT 389 bp, KI 223 bp) transgenes. WT, wild type; HTZ, heterozygous. ‘No DNA’ is a negative control. d, Immunofluorescence on pancreatic sections from control (Ctrl) or β-only mice 4 wpa. INS, insulin (red); GCG, green (left); SST, green (middle); PPY, green (right). Scale bars, 20 µm. e, Quantification of GCG+, SST+ and PPY+ cells in Ctrl (n = 4) or DT (n = 5) mice. GCG+ and SST+ cells were scored in the dorsal pancreas; PPY+ cells were scored in the ventral pancreas. Statistical tests: two-way ANOVA. P values for Ctrl vs DT: GCG+, ****P < 0.0001; SST+, **P = 0.0012; PPY+, ****P < 0.0001. f, qPCR of Gcg, Sst and Ppy on isolated islets of Ctrl (n = 10) and β-only mice at 1 wpa (n = 4), 2 wpa (n = 8) and 4 wpa (n = 7). Data are normalized to housekeeping genes (Gapdh and β-actin) and shown as relative hormone expression to Ctrl. P values for Gcg: 1 wpa vs 0 wpa, ***P = 0.0001; 2 wpa vs 0 wpa, ***P = 0.0001; 4 wpa vs 0 wpa, ****P = 0.00005; Sst: 1 wpa vs 0 wpa, ***P = 0.0001; 2 wpa vs 0 wpa, **P = 0.007; 4 wpa vs 0 wpa, ****P = 0.00005; Ppy: 1 wpa vs 0 wpa, P = 0.056; 2 wpa vs 0 wpa, ***P = 0.0001; 4 wpa vs 0 wpa, ****P = 0.00005. NS, not significant. g, Pancreatic insulin content of Ctrl (n = 10) and DT (n = 10) at 4 wpa. P value Ctrl vs DT, P = 0.63. All data are shown as mean ± s.e.m. Male and female mice were used for these experiments. Unless otherwise indicated, P values are from two-tailed Mann–Whitney tests. NS, not significant.