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. 2016 May 10;5(7):437–448. doi: 10.1016/j.molmet.2016.05.001

Figure 1.

Figure 1

Tamoxifen-pulsed linage tracing of regenerated β-cells. (A) Tamoxifen-inducible labeling of β-cells in mice. The transgene MIP-CreERT2 expresses exclusively in the insulin-producing β-cells a Cre recombinase fused with ERT2, a ligand-binding domain of mutated estrogen receptor. Tamoxifen binds the CreERT2 protein and transports it into the nucleus, where it excises the loxP-flanked stop codon in the Rosa26 knock-in locus and activates the reporter gene (LacZ or tdTomato) expression irreversibly and heritably. (B) X-gal staining (blue) of pancreas from [MIP-CreERT2; Rosa26-LacZ] mice without tamoxifen treatment (left), after 3-week release from the subcutaneously implanted tamoxifen pellet and 1-week washout (middle), and [MIP-CreERT2; Rosa26-LacZ; PANIC-ATTAC] mice after 3-week tamoxifen release, 1-week washout, dimerizer-induced β-cell apoptosis, and 2-month autonomous regeneration (the ATTAC challenge, right). Paraffin sections of the X-gal stained pancreas were counterstained with nuclear fast red (NFR, red). Scale bar: 50 μm. (C) Immunofluorescence of Cre recombinase on pancreas sections from MIP-CreERT2 mice without tamoxifen treatment, after 1-week tamoxifen release, after 3-week tamoxifen release and 1-week washout, and after 3-week tamoxifen release and 4-month washout, as indicated. Top panels: Cre (red) signal only. Bottom panels: Cre merged with insulin (green) and DAPI (blue) signals. Arrowheads: the nuclear Cre signal co-localized with DAPI (purple). Scale bar: 100 μm. (D–F) [MIP-CreERT2; Rosa26-LacZ; PANIC-ATTAC] and [MIP-CreERT2; Rosa26-Tomato; PANIC-ATTAC] mice were subjected to 3-week tamoxifen release and 4-month washout. Before (left) or after (right) the ATTAC challenge, (D) X-gal (blue) staining of pancreas from the Rosa26-LacZ mice. Scale bars: 50 μm. (E) Immunofluorescence of tdTomato (red) and insulin (green) on pancreas sections from the Rosa26-Tomato mice. Top panels: Tomato signal only. Bottom panels: Tomato merged with insulin and DAPI (blue). Arrowheads: Tomato−insulin+ cells (green). Scale bar: 100 μm. (F) In each mouse from (E) the total numbers of Tomato−insulin+ cells and insulin+ cells were counted and calculated for percentage. Data are presented as mean ± SEM. Dots: individual mice. *P < 0.05 by unpaired t-test.