Evaluation of approaches to generation of tissue-specific knock-in mice.

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.