Hepatomas with activating Ctnnb1 mutations in 'Ctnnb1-deficient' livers: a tricky aspect of a conditional knockout mouse model

Conditional knockout mice, based on the Cre-loxP system, are a widely used model for examining organ-specific gene functions. To date, efficient hepatocyte-specific knockout has been reported in many different models, but little attention has been paid to the long-term stability of the recombination efficiency. In the present study, we characterized Alb-Cre;Ctnnb1(flox/flox) 'hepatocyte-specific Ctnnb1 knockout' mice of different ages to test whether efficient recombination is maintained over time. At 2 months of age, the knockout mouse livers achieved efficient deletions of beta-catenin in hepatocytes. However, as the mice aged, the reappearance and expansion of beta-catenin-expressing hepatocytes were observed. In 1-year-old mice, a significant proportion of the pericentral hepatocytes in the knockout mouse livers were replaced with beta-catenin-positive hepatocytes, whereas the periportal hepatocytes mostly remained beta-catenin-negative. Furthermore, most of the 1-year-old mice spontaneously developed hepatocellular adenomas and carcinomas that were positive for beta-catenin and overexpressed glutamine synthetase and Slc1a2, both of which are hallmarks of active beta-catenin signaling. Sequencing analysis revealed that the Ctnnb1 alleles were not inactivated but had activating mutations in these tumors. The present study suggests that recombination efficiency should be carefully examined when hepatocyte-specific knockout mice of different ages are analyzed. In addition, illegitimate deletion mutations should be recognized as potential adverse effects of the Cre-loxP system.