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Hepatomas with activating Ctnnb1 mutations in 'Ctnnb1-deficient' livers: a tricky aspect of a conditional knockout
Shigeki Sekine1, Reiko Ogawa, Yae Kanai
1Pathology Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo, Japan 104-0045. ssekine@ncc.go.jp
Abstract:
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;Ctnnb1flox/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 β-catenin in hepatocytes. However, as the mice aged, the reappearance and expansion of β-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 β-catenin-positive hepatocytes, whereas the periportal hepatocytes mostly remained β-catenin-negative. Furthermore, most of the 1-year-old mice spontaneously developed hepatocellular adenomas and carcinomas that were positive for β-catenin and overexpressed glutamine synthetase and Slc1a2, both of which are hallmarks of active β-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.
Insights
Hepatocyte-specific knockout mice using the Cre-loxP system show declining gene deletion efficiency over time. Aged mice develop tumors with reactivated beta-catenin signaling due to mutations, not recombination loss.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Conditional knockout mice, particularly using the Cre-loxP system, are vital for studying organ-specific gene functions.
- Efficient hepatocyte-specific gene knockout models are widely used, but long-term recombination stability is often overlooked.
Purpose of the Study:
- To evaluate the long-term stability of recombination efficiency in hepatocyte-specific Ctnnb1 (beta-catenin) knockout mice.
- To investigate the consequences of declining recombination efficiency on liver tissue and tumor development over time.
Main Methods:
- Utilized Alb-Cre;Ctnnb1flox/flox mice to create hepatocyte-specific beta-catenin knockouts.
- Analyzed liver tissues from mice of different ages (2 months and 1 year) to assess recombination efficiency.
- Performed sequencing to identify genetic alterations in tumors.
Main Results:
- Efficient beta-catenin deletion in hepatocytes was observed at 2 months of age.
- Aged mice (1 year) showed reappearance and expansion of beta-catenin-expressing hepatocytes, particularly in pericentral regions.
- Hepatocellular adenomas and carcinomas developed in aged knockout mice, characterized by beta-catenin positivity, glutamine synthetase/Slc1a2 overexpression, and activating Ctnnb1 mutations.
Conclusions:
- Long-term stability of Cre-loxP-mediated recombination in hepatocyte-specific knockout models requires careful age-dependent evaluation.
- Activating mutations in the target gene can occur, leading to tumor formation, and should be considered as a potential artifact of the Cre-loxP system.
