The glucocorticoid receptor (Gr, encoded by the gene Grl1) controls transcription of target genes both directly by interaction with DNA regulatory elements and indirectly by cross-talk with other transcription factors. In response to various stimuli, including stress, glucocorticoids coordinate metabolic, endocrine, immune and nervous system responses and ensure an adequate profile of transcription. In the brain, Gr has been proposed to modulate emotional behaviour, cognitive functions and addictive states. Previously, these aspects were not studied in the absence of functional Gr because inactivation of Grl1 in mice causes lethality at birth (F.T., C.K. and G.S., unpublished data). Therefore, we generated tissue-specific mutations of this gene using the Cre/loxP -recombination system. This allowed us to generate viable adult mice with loss of Gr function in selected tissues. Loss of Gr function in the nervous system impairs hypothalamus-pituitary-adrenal (HPA)-axis regulation, resulting in increased glucocorticoid (GC) levels that lead to symptoms reminiscent of those observed in Cushing syndrome. Conditional mutagenesis of Gr in the nervous system provides genetic evidence for the importance of Gr signalling in emotional behaviour because mutant animals show an impaired behavioural response to stress and display reduced anxiety.

Insulin receptors (IRs) and insulin signaling proteins are widely distributed throughout the central nervous system (CNS). To study the physiological role of insulin signaling in the brain, we created mice with a neuron-specific disruption of the IR gene (NIRKO mice). Inactivation of the IR had no impact on brain development or neuronal survival. However, female NIRKO mice showed increased food intake, and both male and female mice developed diet-sensitive obesity with increases in body fat and plasma leptin levels, mild insulin resistance, elevated plasma insulin levels, and hypertriglyceridemia. NIRKO mice also exhibited impaired spermatogenesis and ovarian follicle maturation because of hypothalamic dysregulation of luteinizing hormone. Thus, IR signaling in the CNS plays an important role in regulation of energy disposal, fuel metabolism, and reproduction.

Deletion of the promoter and the first exon of the DNA polymerase beta gene (pol beta) in the mouse germ line results in a lethal phenotype. With the use of the bacteriophage-derived, site-specific recombinase Cre in a transgenic approach, the same mutation can be selectively introduced into a particular cellular compartment-in this case, T cells. The impact of the mutation on those cells can then be analyzed because the mutant animals are viable.

Mice that lack all beta1-class integrins in neurons and glia die prematurely after birth with severe brain malformations. Cortical hemispheres and cerebellar folia fuse, and cortical laminae are perturbed. These defects result from disorganization of the cortical marginal zone, where beta1-class integrins regulate glial endfeet anchorage, meningeal basement membrane remodeling, and formation of the Cajal-Retzius cell layer. Surprisingly, beta1-class integrins are not essential for neuron-glia interactions and neuronal migration during corticogenesis. The phenotype of the beta1-deficient mice resembles pathological changes observed in human cortical dysplasias, suggesting that defective integrin-mediated signal transduction contributes to the development of some of these diseases.

We have developed a method of specifically modifying the mammalian genome in vivo. This procedure comprises heritable tissue-specific and site-specific DNA recombination as a function of recombinase expression in transgenic mice. Transgenes encoding the bacteriophage P1 Cre recombinase and the loxP-flanked fi-galactosidase gene were used to generate transgenic mice. Genomic DNA from doubly transgenic mice exhibited tissue-specific DNA recombination as a result of Cre expression. Further characterization revealed that this process was highly efficient at distinct chromosomal integration sites. These studies also imply that Cre-mediated recombination provides a heritable marker for mitoses following the loss of Cre expression. This transgene-recombination system permits unique approaches to in vivo studies of gene function within experimentally defmed spatial and temporal boundaries.Bacteriophage P1 encodes the 38-kDa Cre recombinase that catalyzes site-specific DNA recombination between 34-basepair (bp) repeats termed loxP (1). Cre is a member of the integrase family of recombinases. These enzymes recognize specific nucleotide sequences and function through a transient DNA-protein covalent linkage (reviewed in refs. 2 and 3). Cre activity appears mechanistically identical to that of yeast FLP recombinase and can function in vitro in the absence of high-energy cofactors, topoisomerase activity, and DNA replication (4, 5). In Cre-mediated recombination, resultant DNA structures are dependent upon the orientation of loxP sites. Direct repeats of loxP dictate an excision of intervening sequences whereas inverted repeats specify inversion (4). Cre and FLP have been shown to mediate site-specific DNA recombination in tissue-cultured eukaryotic cells, Drosophila, and transgenic plants (6)(7)(8)(9)(10)(11)(12).With the aim of applying Cre recombinase function to molecular studies of normal and abnormal mammalian physiology, we sought to generate a transgenic mouse system that would establish whether Cre could effectively mediate chromosomal DNA recombination. As a foundation for future applications, we devised a nondeleterious transgene strategy that would provide an assessment of the efficiency, position dependence, and heritability of Cre-mediated chromosomal DNA recombination in mammals.