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Published on: June 15, 2017
Evaluation of approaches to generation of tissue-specific knock-in mice
Jose R Bayascas1, Kei Sakamoto, Laura Armit
1MRC Protein Phosphorylation Unit and School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom. j.bayascas@dundee.ac.uk
Abstract:
We explored three approaches to create tissue-specific knock-in mice by generating knock-in mice in which a substrate-docking site of the PDK1 protein kinase was ablated in Cre-expressing tissues in a way that prevented activation of one of its substrates, p70 ribosomal S6 kinase (S6K), but not another (protein kinase B (PKB)). Employing two of the approaches, termed the "heterozygous" and "minigene" methods, we generated mice in which Cre-expressing skeletal and cardiac muscle produced the mutant rather than wild type PDK1. Consistent with this, injection of these mice with insulin only induced activation of PKB but not S6K in muscle tissues. We have also demonstrated that insulin-stimulated glucose uptake proceeds normally in knock-in mice, consistent with the notion that PKB mediates this process. In contrast to conditional knock-out of PDK1 in muscle, the knock-in mice did not develop dilated cardiomyopathy, suggesting that PKB plays a key role in protecting mice from heart failure. The third knock-in strategy that was evaluated, termed the "inversion" method, did not proceed with high efficiency. We discuss the merits and disadvantages of each of the conditional knock-in approaches, along with the applications for which they may be most suited, and suggest how they could be further refined.
Insights
Researchers developed conditional knock-in mice to study PDK1 protein kinase. This method selectively inactivated S6K, not PKB, in specific tissues, revealing PKB
Area of Science:
- Molecular Biology
- Genetics
- Physiology
Background:
- PDK1 protein kinase is crucial for cell signaling pathways.
- Understanding PDK1's role in specific tissues requires precise genetic manipulation.
- Conditional knock-in models allow for tissue-specific gene function studies.
Purpose of the Study:
- To develop and evaluate conditional knock-in mouse models for studying PDK1 function.
- To investigate the distinct roles of PDK1 substrates, S6K and PKB, in insulin signaling and cardiac function.
- To assess the potential of these models for understanding tissue-specific signaling.
Main Methods:
- Generated conditional knock-in mice by ablating the PDK1 substrate-docking site in Cre-expressing tissues.
- Utilized heterozygous and minigene methods for generating knock-in models.
- Administered insulin to knock-in mice and analyzed PKB and S6K activation, and glucose uptake.
Main Results:
- Successfully generated knock-in mice where skeletal and cardiac muscle produced mutant PDK1, preventing S6K activation but not PKB activation.
- Insulin-stimulated glucose uptake remained normal in knock-in mice, supporting PKB's role.
- Knock-in mice did not develop dilated cardiomyopathy, unlike conditional knock-out models, suggesting PKB's protective role in heart failure.
Conclusions:
- Conditional knock-in strategies, particularly the heterozygous and minigene methods, are effective for studying tissue-specific kinase function.
- PKB plays a critical role in insulin-mediated glucose uptake and cardiac protection.
- These models offer valuable insights into the differential roles of PDK1 substrates in physiological processes.
