Figure 1.

Generation and characterization of MybER transgenic mice. (a) A fusion of mouse Myb to a mutant ERT2 estrogen receptor ligand-binding domain was generated under the control of the intestinal Gpa33-specific antigen promoter. (b) Model of tamoxifen (4OHT)-activation of the MybER protein to reveal the DNA-binding domain and transactivation domain (T/A) of Myb. (c) Insertion of the transgene was confirmed by Southern blotting using a Myb probe. (d) MybER protein expression (black arrow) was confirmed by western blotting with an anti-ERα antibody in the colonic crypt lysates from a WT and a MybER mouse, respectively, exposed to tamoxifen for 2 weeks. Pan actin was used a protein-loading control (gray arrow). (e) Colonic crypt lysates were also probed for Myb expression in parallel with CRC cell line CT26 with a cocktail to anti-Myb antibodies that binds amino-terminal to the fusion junction point of Myb in the MybER protein to detect a protein of the predicted size. (f) Organoid cultures derived from colonic crypts seeded in Matrigel in the presence of 0.1 μM 4OHT show increased colony size in MybER cultures. (g) Quantification of colonies formed/plated crypt nests and overall increased growth as assessed by the MTT assay. Cultures were grown for 7–10 days in the presence of 0.1 μM 4OHT before harvesting. Data presented for individual samples are shown; n>5; mean ± s.e.m.; *P<0.05, two sided t-test. Primary organoids formed in presence of 4OHT were then dissociated and secondary organoids generated ± 4OHT were scored after 5 days. (h) ChIP assay showing the occupancy of Lgr5 promoter by the fusion protein MybER following activation by 4OHT. ChIP was quantified by qRT–PCR and data were normalized for Gapdh promoter (n=4 independent experiments in triplicate). Data presented for individual samples (n>4) with mean ± s.e.m.; *P<0.05, ordinary one-way ANOVA.