Frieder Schwenk

Inducible gene targeting in mice.

A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. The method uses an interferon-responsive promoter to control the expression of Cre recombinase. Here, Cre was used to delete a segment of the DNA polymerase beta gene flanked by IoxP recombinase recognition sites. Deletion was complete in liver and nearly complete in lymphocytes within a few days, whereas partial deletion was obtained in other tissues. This method can be used for the inducible inactivation of any other gene in vivo.

Enhanced PIP3 signaling in POMC neurons causes KATP channel activation and leads to diet-sensitive obesity

Leptin and insulin have been identified as fuel sensors acting in part through their hypothalamic receptors to inhibit food intake and stimulate energy expenditure. As their intracellular signaling converges at the PI3K pathway, we directly addressed the role of phosphatidylinositol3,4,5-trisphosphate-mediated (PIP3-mediated) signals in hypothalamic proopiomelanocortin (POMC) neurons by inactivating the gene for the PIP3 phosphatase Pten specifically in this cell type. Here we show that POMC-specific disruption of Pten resulted in hyperphagia and sexually dimorphic diet-sensitive obesity. Although leptin potently stimulated Stat3 phosphorylation in POMC neurons of POMC cell-restricted Pten knockout (PPKO) mice, it failed to significantly inhibit food intake in vivo. POMC neurons of PPKO mice showed a marked hyperpolarization and a reduction in basal firing rate due to increased ATP-sensitive potassium (KATP) channel activity. Leptin was not able to elicit electrical activity in PPKO POMC neurons, but application of the PI3K inhibitor LY294002 and the KATP blocker tolbutamide restored electrical activity and leptin-evoked firing of POMC neurons in these mice. Moreover, icv administration of tolbutamide abolished hyperphagia in PPKO mice. These data indicate that PIP3-mediated signals are critical regulators of the melanocortin system via modulation of KATP channels.

Central insulin action regulates peripheral glucose and fat metabolism in mice

Insulin resistance is a hallmark of type 2 diabetes, and many insights into the functions of insulin have been gained through the study of mice lacking the IR. To gain a better understanding of the role of insulin action in the brain versus peripheral tissues, we created 2 mouse models with inducible IR inactivation, 1 in all tissues including brain (IRDeltawb), and 1 restricted to peripheral tissues (IRDeltaper). While downregulation of IR expression resulted in severe hyperinsulinemia in both models, hyperglycemia was more pronounced in IRDeltawb mice. Both strains displayed a dramatic upregulation of hepatic leptin receptor expression, while only IRDeltaper mice displayed increased hepatic Stat3 phosphorylation and Il6 expression. Despite a similar reduction in IR expression in white adipose tissue (WAT) mass in both models, IRDeltawb mice had a more pronounced reduction in WAT mass and severe hypoleptinemia. Leptin replacement restored hepatic Stat3 phosphorylation and normalized glucose metabolism in these mice, indicating that alterations in glucose metabolism occur largely as a consequence of lipoathrophy upon body-wide IR deletion. Moreover, chronic intracerebroventricular insulin treatment of control mice increased fat mass, fat cell size, and adipose tissue lipoprotein lipase expression, indicating that CNS insulin action promotes lipogenesis. These studies demonstrate that central insulin action plays an important role in regulating WAT mass and glucose metabolism via hepatic Stat3 activation.

Reversible gene knockdown in mice using a tight, inducible shRNA expression system

RNA interference through expression of short hairpin (sh)RNAs provides an efficient approach for gene function analysis in mouse genetics. Techniques allowing to control time and degree of gene silencing in vivo, however, are still lacking. Here we provide a generally applicable system for the temporal control of ubiquitous shRNA expression in mice. Depending on the dose of the inductor doxycycline, the knockdown efficiency reaches up to 90%. To demonstrate the feasibility of our tool, a mouse model of reversible insulin resistance was generated by expression of an insulin receptor (Insr)-specific shRNA. Upon induction, mice develop severe hyperglycemia within seven days. The onset and progression of the disease correlates with the concentration of doxycycline, and the phenotype returns to baseline shortly after withdrawal of the inductor. On a broad basis, this approach will enable new insights into gene function and molecular disease mechanisms.

Single copy shRNA configuration for ubiquitous gene knockdown in mice

RNA interference through the expression of small hairpin RNA (shRNA) molecules has become a very promising tool in reverse mouse genetics as it may allow inexpensive and rapid gene function analysis in vivo. However, the prerequisites for ubiquitous and reproducible shRNA expression are not well defined. Here we show that a single copy shRNA-transgene can mediate body-wide gene silencing in mice when inserted in a defined locus of the genome. The most commonly used promoters for shRNA expression, H1 and U6, showed a comparably broad activity in this configuration. Taken together, the results define a novel approach for efficient interference with expression of defined genes in vivo. Moreover, we provide a rapid strategy for the production of gene knockdown mice combining recombinase mediated cassette exchange and tetraploid blastocyst complementation approaches.

The histone 3 lysine 4 methyltransferase, Mll2, is only required briefly in development and spermatogenesis

BackgroundHistone methylation is thought to be central to the epigenetic mechanisms that maintain and confine cellular identity in multi-cellular organisms. To examine epigenetic roles in cellular homeostasis, we conditionally mutated the histone 3 lysine 4 methyltransferase, Mll2, in embryonic stem (ES) cells, during development and in adult mice using tamoxifen-induced Cre recombination.ResultsIn ES cells, expression profiling unexpectedly revealed that only one gene, Magoh2, is dependent upon Mll2 and few other genes were affected. Loss of Mll2 caused loss of H3K4me3 at the Magoh2 promoter and concomitant gain of H3K27me3 and DNA methylation. Hence Mll2, which is orthologous to Drosophila Trithorax, is required to prevent Polycomb-Group repression of the Magoh2 promoter, and repression is further accompanied by DNA methylation. Early loss of Mll2 in utero recapitulated the embryonic lethality found in Mll2-/- embryos. However, loss of Mll2 after E11.5 produced mice without notable pathologies. Hence Mll2 is not required for late development, stem cells or homeostasis in somatic cell types. However it is required in the germ cell lineage. Spermatogenesis was lost upon removal of Mll2, although spermatogonia A persisted.ConclusionThese data suggest a bimodal recruit and maintain model whereby Mll2 is required to establish certain epigenetic decisions during differentiation, which are then maintained by redundant mechanisms. We also suggest that these mechanisms relate to the epigenetic maintenance of CpG island promoters.

Residual MHC class II expression on mature dendritic cells and activated B cells in RFX5-deficient mice

Patients with major histocompatibility complex class II (MHC-II) deficiency are known to carry mutations in either the RFX complex or the trans-activator CIITA. While the pivotal role of CIITA for MHC-II gene transcription is supported by the essential absence of MHC-II molecules in CIITA-deficient mice, we demonstrate here that RFX5-/- mice retain expression of MHC-II in thymic medulla, mature dendritic cells, and activated B cells. Nevertheless, RFX5-/- mice develop a severe immunodeficiency due to the lack of MHC-II in thymic cortex, failure of positive selection of CD4+ T cells, and absence of MHC-II on resting B cells and resident or IFNgamma-activated macrophages. This differential requirement for CIITA and RFX5 in subsets of antigen-presenting cells may be specific for the mouse; it may, however, also exist in humans without having been noticed so far.

A Genetic Switch for Epilepsy in Adult Mice

Premature death from seizures afflicts gene-targeted mice expressing the Q/R site-unedited glutamate receptor subunit GluR-B(Q) of AMPA receptors in central neurons. Early seizure-related death has now been circumvented by a genetic switch that restricts GluR-B(Q) expression to forebrain principal neurons from postnatal stages onward, prominently in hippocampus and striatum and less so in cortex and amygdala. When switched on, functional receptor incorporation of GluR-B(Q) could be demonstrated by imaging evoked AMPA channel-mediated spinous Ca2+ transients in CA1 pyramidal cells. Sustained GluR-B(Q) expression in adult mice led to smaller excitatory postsynaptic responses in the CA1 region with unchanged presynaptic fiber excitability. Notably, despite the smaller excitatory response, the CA1 cells exhibited a reduced population spike threshold, which might underlie the spontaneous manifestations of epilepsy, including myocloni and generalized seizures with limbic components, observed by synchronous video monitoring and electroencephalographic recordings. No neuropathological symptoms developed when GluR-B(Q) expression was restricted to only hippocampal neurons. Our results show that seizure susceptibility is triggered by GluR-B(Q) expression also in the adult brain and that circuit hyperexcitability is not an immediate consequence of GluR-B(Q) but requires yet unknown downstream events, likely to be induced by non-Hebbian plasticity from Ca2+-permeable AMPA channels in principal neurons.

Advances in gene targeting methods

Gene targeting in embryonic stem cells is commonly used for gene inactivation and the generation of mouse mutants. The combined use of methods for site-specific and homologous DNA recombination expands the potential of gene targeting in embryonic stem cells considerably and offers the opportunity of conditional gene targeting in mice.

Hybrid Embryonic Stem Cell-Derived Tetraploid Mice Show Apparently Normal Morphological, Physiological, and Neurological Characteristics

ABSTRACT 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 B6129S6F1 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.