Characterization and Validation of Cre-Driver Mouse Lines.

Conditional gene manipulations in mice are increasingly popular strategies in biomedical research. These approaches rely on the production of conditional genetically engineered mutant mouse (GEMM) lines with mutations in protein-encoding genes. These conditional GEMMs are then bred with one or several transgenic mouse lines expressing a site-specific recombinase, most often the Cre recombinase, in a tissue-specific manner. Conditional GEMMs can only be exploited if Cre transgenic mouse lines are available to generate somatic mutations, and thus the number of Cre transgenic lines has significantly increased over the last 15 years. Once produced, these transgenic lines must be validated for reliable, efficient, and specific Cre expression and Cre-mediated recombination. In this overview, the minimum level of information that is ideally required to validate a Cre-driver transgenic line is first discussed. The vagaries associated with validation procedures are considered next, and some solutions are proposed to assess the expression and activity of constitutive or inducible Cre recombinase before undertaking extensive breeding experiments and exhaustive phenotyping. Curr. Protoc. Mouse Biol. 1:1-15. © 2011 by John Wiley & Sons, Inc.

Mouse Breeding and Colony Management.

The possibility to genetically modify the mouse genome has enabled the creation of numerous lines of genetically engineered mouse models (GEMMs). As a result, the demand for housing space in research facilities is increasing. Knowledge of the basis of mouse reproduction and of the methods to handle colonies of GEMMs is therefore mandatory to efficiently populate facilities. The mouse has a short generation period, produces large progenies, and can breed all year round. However, environmental parameters (bedding, diet, cage type, temperature, hygrometry, light, noise, and sanitary status) strongly influence the breeding efficiency and experimental data, and must be tightly controlled. Efficient GEMM colony management requires adequate recording of breeding and proper identification and genotyping of animals. Various mating types and breeding schemes can be used, depending on the type of studies conducted. The recent development of assisted reproduction methods helps circumvent some of the issues faced with those lines especially difficult to breed. Curr. Protoc. Mouse Biol. 1:239-264. © 2011 by John Wiley & Sons, Inc.

Site-specific recombinases for manipulation of the mouse genome.

Site-specific recombination systems are widespread and popular tools for all scientists interested in manipulating the mouse genome. In this chapter, we focus on the use of site-specific recombinases (SSR) to unravel the function of genes of the mouse. In the first part, we review the most commonly used SSR, Cre and Flp, as well as the newly developed systems such as Dre and PhiC31, and we present the inducible SSR systems. As experience has shown that these systems are not as straightforward as expected, particular attention is paid to facts and artefacts associated with their production and applications to study the mouse genome. In the next part of this chapter, we illustrate new applications of SSRs that allow engineering of the mouse genome with more and more precision, including the FLEX and the RMCE strategies. We conclude and suggest a workflow procedure that can be followed when using SSR to create your mouse model of interest. Together, these strategies and procedures provide the basis for a wide variety of studies that will ultimately lead to the analysis of the function of a gene at the cellular level in the mouse.

Synthesis and evaluation of anilinohexafluoroisopropanols as activators/modulators of LXRalpha and beta.

A series of branched and unbranched anilinohexafluoroisopropanols related to the known sulfonamide T0901317 were prepared and evaluated as activators/modulators of both LXRalpha and LXRbeta. A structure-activity relationship was established and compounds with high potency on both the receptors were identified. Many compounds showed a tendency toward selectivity for LXRbeta versus LXRalpha. Several analogues were evaluated for effects on plasma lipoprotein levels in mice. A few of these significantly raised HDL-cholesterol levels in plasma but showed markedly different effects on liver triglyceride content, suggesting that this series may yield candidates with improved efficacy/safety profiles compared to existing molecules.

Neuregulin 1-erbB signaling is necessary for normal myelination and sensory function.

To investigate the role of erbB signaling in the interactions between peripheral axons and myelinating Schwann cells, we generated transgenic mice expressing a dominant-negative erbB receptor in these glial cells. Mutant mice have delayed onset of myelination, thinner myelin, shorter internodal length, and smaller axonal caliber in adulthood. Consistent with the morphological defects, transgenic mice also have slower nerve conduction velocity and defects in their responses to mechanical stimulation. Molecular analysis indicates that erbB signaling may contribute to myelin formation by regulating transcription of myelin genes. Analysis of sciatic nerves showed a reduction in the levels of expression of myelin genes in mutant mice. In vitro assays revealed that neuregulin-1 (NRG1) induces expression of myelin protein zero (P0). Furthermore, we found that the effects of NRG1 on P0 expression depend on the NRG1 isoform used. When NRG1 is presented to Schwann cells in the context of cell-cell contact, type III but not type I NRG1 regulates P0 gene expression. These results suggest that disruption of the NRG1-erbB signaling pathway could contribute to the pathogenesis of peripheral neuropathies with hypomyelination and neuropathic pain.

Insulin receptor substrate-2 deficiency impairs brain growth and promotes tau phosphorylation.

Insulin resistance and diabetes might promote neurodegenerative disease, but a molecular link between these disorders is unknown. Many factors are responsible for brain growth, patterning, and survival, including the insulin-insulin-like growth factor (IGF)-signaling cascades that are mediated by tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. Irs2 signaling mediates peripheral insulin action and pancreatic beta-cell function, and its failure causes diabetes in mice. In this study, we reveal two important roles for Irs2 signaling in the mouse brain. First, disruption of the Irs2 gene reduced neuronal proliferation during development by 50%, which dissociated brain growth from Irs1-dependent body growth. Second, neurofibrillary tangles containing phosphorylated tau accumulated in the hippocampus of old Irs2 knock-out mice, suggesting that Irs2 signaling is neuroprotective. Thus, dysregulation of the Irs2 branch of the insulin-Igf-signaling cascade reveals a molecular link between diabetes and neurodegenerative disease.

Physiological and retinoid-induced proliferations of epidermis basal keratinocytes are differently controlled.

To investigate the roles of retinoic acid (RA) receptors (RARs) in the physiology of epidermis that does not express RAR beta, conditional spatio-temporally controlled somatic mutagenesis was used to selectively ablate RAR alpha in keratinocytes of RAR gamma-null mice. Keratinocyte proliferation was maintained in adult mouse epidermis lacking both RAR alpha and RAR gamma, as well as in RAR beta-null mice. All RAR-mediated signalling pathways are therefore dispensable in epidermis for homeostatic keratinocyte renewal. However, topical treatment of mouse skin with selective retinoids indicated that RXR/RAR gamma heterodimers, in which RXR transcriptional activity was subordinated to that of its RAR gamma partner, were required for retinoid-induced epidermal hyperplasia, whereas RXR homodimers and RXR/RAR alpha heterodimers were not involved. RA-induced keratinocyte proliferation was studied in mutant mice in which RXR alpha, RXR alpha and RAR alpha, RAR gamma, or RXR alpha and RAR gamma genes were specifically disrupted in either basal or suprabasal keratinocytes. We demonstrate that the topical retinoid signal is transduced by RXR alpha/RAR gamma heterodimers in suprabasal keratinocytes, which, in turn, stimulate proliferation of basal keratinocytes via a paracrine signal that may be heparin-binding EGF-like growth factor.