Evaluation of Approaches to Generation of Tissue-specific Knock-in Mice

Jose R Bayascas,Kei Sakamoto,Laura Armit,J Simon C Arthur,Dario R Alessi

doi:10.1074/jbc.m606789200

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.

Highlights

For the heterozygous method described previously, mice in which the wild type PDK1 allele was flanked with LoxP sites (PDK1fl/fl) [20] were bred with PDK1L155E/ϩ knock-in mice [14] to generate PDK1fl/L155E animals (Fig. 2A), which were in turn crossed with transgenic mice expressing the Cre recombinase under the Mck promoter
The results presented in this study indicate that both the heterozygous and minigene methodology, in combination with MckCre transgenic mice, can be successfully deployed to generate mice expressing mutant PDK1[L155E] rather than wild type PDK1 in heart and muscle tissues
We had previously found that conditional knock-out mice lacking PDK1 in heart muscle in which neither PKB or S6 kinase (S6K) are activated develop dilated cardiomyopathy and die of heart failure at 5–11 weeks of age [24]

Summary

Introduction

R. A.), and by the pharmaceutical companies supporting the Division of Signal Transduction Therapy (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck & Co. Inc., Merck KGaA, and Pfizer). The costs of publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. 1 Supported by a long-term fellowship from the European Molecular Biology Organization as well as a Marie Curie fellowship. To whom correspondence should be addressed: MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, United Kingdom. Tel.: 44-1382-384-241; Fax: 44-1382-223-778; E-mail: j.bayascas@ dundee.ac.uk

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