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. 2014 May 29;111(1):132–138. doi: 10.1038/bjc.2014.275

Figure 1.

Figure 1

Deletion of β-catenin in β-catenin-activated mouse liver tumours. (A) Schematic representation of the Ctnnb1 gene in livers of transgenic mice. Expression of the modified Cre protein MerCreMer is under the control of the liver-specific transthyretin (TTR) promoter. In the presence of the tamoxifen metabolite 4-hydroxytamoxifen (4-OHT), Cre recombinase, which is flanked by modified ligand-binding domains of the mouse oestrogen receptor (Mer), is released from its chaperones (heat shock protein 90, Hsp90) and targets loxP sites flanking the exons 3 and 6 within the Ctnnb1 gene. Cre-mediated recombination results in a deleted allele encoding a non-functional β-catenin protein. (B) Schematic delineation of experimental setup: mice received a single intraperitoneal (i.p.) injection of N-DEN (90 μg g−1 body weight) at 6 weeks of age. Subsequently, mice were continuously fed a diet containing 0.05% PB to select for Ctnnb1-mutated hepatomas. After a treatment-free period of 1 week, deletion of the Ctnnb1 gene in KO mice was achieved by i.p. injection of 1.5 mg tamoxifen for 5 consecutive days. Animals were then killed at different time points after application. (C) PCR analysis of tumour DNA from two representative Ctnnb1 KO and WT mice. The recombined, deleted Ctnnb1 gene was detected exclusively in Cre-expressing KO mice after tamoxifen treatment. Non-recombined Ctnnb1loxP/loxP was detected in both Ctnnb1 KO and WT mice but with lower amounts in Ctnnb1 KO mice. The Apc gene was amplified as a reference gene. (D) Immunostaining for GS, a marker for β-catenin activation. Ctnnb1-mutated tumours from WT animals show uniform expression of GS (left images). After KO of Ctnnb1, GS-negative hepatoma cells are observed within the tumours. The number of residual GS-positive cells varies between different tumours. (E) Tumours from Ctnnb1 WT and KO mice double stained for β-catenin and BrdU. β-catenin is present at the cell membrane of all tumour cells from WT mice, whereas only single cells possess β-catenin in tumour tissue from a respective KO animal.