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Biomedical subjects

Ralf Kühn

Publications and source records attributed to Ralf Kühn.

8 recordsLinked to original sources
PubMed2003

Limbic corticotropin-releasing hormone receptor 1 mediates anxiety-related behavior and hormonal adaptation to stress.

Corticotropin-releasing hormone (CRH) is centrally involved in coordinating responses to a variety of stress-associated stimuli. Recent clinical data implicate CRH in the pathophysiology of human affective disorders. To differentiate the CNS pathways involving CRH and CRH receptor 1 (Crhr1) that modulate behavior from those that regulate neuroendocrine function, we generated a conditional knockout mouse line (Crhr1(loxP/loxP)Camk2a-cre) in which Crhr1 function is inactivated postnatally in anterior forebrain and limbic brain structures, but not in the pituitary. This leaves the hypothalamic-pituitary-adrenocortical (HPA) system intact. Crhr1(loxP/loxP)Camk2a-cre mutants showed reduced anxiety, and the basal activity of their HPA system was normal. In contrast to Crhr1 null mutants, conditional mutants were hypersensitive to stress corticotropin and corticosterone levels remained significantly elevated after stress. Our data clearly show that limbic Crhr1 modulates anxiety-related behavior and that this effect is independent of HPA system function. Furthermore, we provide evidence for a new role of limbic Crhr1 in neuroendocrine adaptation to stress.

Adaptation, Physiological↗
PubMed2003

Rapid generation of inducible mouse mutants.

We have generated an optimized inducible recombination system for conditional gene targeting based on a Cre recombinase-steroid receptor fusion. This configuration allows efficient Cre-mediated recombination in most organs of the mouse upon induction, without detectable background activity. An ES cell line, was established that carries the inducible recombinase and a loxP-flanked lacZ reporter gene. Out of this line, completely ES cell-derived mice were efficiently produced through tetraploid blastocyst complementation, without the requirement of mouse breeding. Our findings provide a new concept allowing the generation of inducible mouse mutants within 6 months, as compared to 14 months using the current protocol.

Animals↗
PubMed2003

Neuron-specific ablation of PDGF-B is compatible with normal central nervous system development and astroglial response to injury.

Members of the PDGF family have multiple roles during embryogenesis and in a variety of pathological situations in the adult. One of the major sites of PDGF-B expression in adult mammals are postmitotic CNS neurons. Combined with reported neurotrophic and neuroprotective effects of exogenously administered PDGFs, this has led to the speculation that PDGF-B may have a role in CNS development, in maintenance, or in response to CNS injury. To test these hypotheses, we developed mice in which PDGF-B was ablated genetically in postmitotic neurons at sites where PDGF-B is normally expressed. We found that these mice develop to adulthood without apparent defects. We demonstrate PDGF-B expression in the postnatal mouse hippocampus and forebrain cortex. We show that neuron-specific knockout of PDGF-B does not influence the astroglial and angiogenic responses to injury in the hippocampus or forebrain cortex. We conclude that the role of neuron-derived PDGF-B remains obscure. A role for neuron-derived PDGF-B, if existing, might be redundant with other CNS growth factors. Alternatively, other and more specific analyses of CNS functions in the normal and injured states will be required to demonstrate such a role.

Animals↗
PubMed2003

Connexin43 is not expressed in principal cells of mouse cortex and hippocampus.

Previous immunofluorescence analyses in mice and rats showed a mainly astrocytic expression of the gap junction protein connexin43 (Cx43) in brain. However, in situ hybridization of murine brain sections suggested strong expression of Cx43 mRNA in hippocampal and cortical pyramidal neurons and Purkinje cells. These findings contrast with recent immunoelectron microscopic studies that excluded prominent Cx43 protein expression in neurons. Both contrasting results could be explained by post-transcriptional control mechanisms. Here we demonstrate by conditional replacement of the Cx43 coding region by a lacZ reporter gene, mimicking transcriptional activity of the Cx43 gene, that Cx43 is not expressed in principal cells of murine brain. This histochemical approach used is not prone to cross-reactivity of mRNA probes or antibodies. Furthermore, we show that in situ hybridization signals, suggested to be specific for Cx43 in mouse neurons, are retained even when the Cx43 coding DNA in neurons is removed by cre-mediated deletion. Our results confirm the previous findings of a mainly astrocytic expression of Cx43 in adult mouse brain and underscore the importance of connexin-deficient mice as controls for in situ hybridization studies. We found no evidence for post-transcriptional control of the Cx43 gene in principal neurons. Thus, the synchronized activity of neuronal networks cannot depend on Cx43 containing gap junctions in these cells.

Animals↗
PubMed2003

Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological, physiological, and neurological characteristics.

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F(1) ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo.

Animals↗
PubMed2002

Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells.

The integrase of the phage PhiC31 recombines an attP site in the phage genome with a chromosomal attB site of its Streptomyces host. We have utilized the integrase-mediated reaction to achieve episomal and genomic deletion of a reporter gene in mammalian cells, and provide the first comparison of its efficiency with other recombinases in a new assay system. This assay demonstrated that the efficiency of PhiC31-integrase is significantly enhanced by the C-terminal, but not the N-terminal, addition of a nuclear localization signal and becomes comparable with that of the widely used Cre/loxP system. Furthermore, we found that the improved FLP recombinase, FLPe, exhibits only 10% recombination activity on chromosomal targets as compared with Cre, whereas the Anabaena derived XisA recombinase is essentially inactive in mammalian cells. These results provide the first demonstration that a nuclear localisation signal and its position within a recombinase can be important for its efficiency in mammalian cells and establish the improved PhiC31-integrase as a new tool for genome engineering.

3T3 Cells↗