RNA Interference in Mice Kühn, R.; Streif, S.; Wurst, W.
Conditional Mutagenesis: An Approach to Disease Models,
2007
178
Book Chapter, Journal Article
Recenzirano
Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for functional genomics in mammalian cells. Furthermore, RNAi holds promise as a simple, fast and cost-effective ...approach to studying mammalian gene function in vivo and as a novel therapeutic approach. This review provides an overview of the progress of RNAi in vivo, with emphasis on systemic/local siRNA delivery, viral shRNA vectors, shRNA vector transgenic mice and conditional systems to control shRNA vectors. Taken together, the data from 80 in vivo studies show that RNAi is a useful tool that offers new opportunities for functional genomics in mice.
During vertebrate limb development, positional information must be specified along three distinct axes. Although much progress has been made in our understanding of the molecular interactions ...involved in anterior-posterior and proximal-distal limb patterning, less is known about dorsal-ventral patterning. The genes Wnt-7a and Lmx-1, which are expressed in dorsal limb ectoderm and mesoderm, respectively, are thought to be important regulators of dorsal limb differentiation. Whether a complementary set of molecules controls ventral limb development has not been clear. Here we report that Engrailed-1, a homeodomain-containing transcription factor expressed in embryonic ventral limb ectoderm, is essential for ventral limb patterning. Loss of Engrailed-1 function in mice results in dorsal transformations of ventral paw structures, and in subtle alterations along the proximal-distal limb axis. Engrailed-1 seems to act in part by repressing dorsal differentiation induced by Wnt-7a, and is essential for proper formation of the apical ectodermal ridge.
Two organizing centres operate at long-range distances within the anterior neural plate to pattern the forebrain, midbrain and hindbrain. Important progress has been made in understanding the ...formation and function of one of these organizing centres, the isthmic organizer, which controls the development of the midbrain and anterior hindbrain. Here we review our current knowledge on the identity, localization and maintenance of the isthmic organizer, as well as on the molecular cascades that underlie the activity of this organizing centre.
Corticotropin-releasing hormone plays an important role in the coordination of various responses to stress. Previous research has implicated both corticotropin-releasing hormone and the serotonergic ...system as causative factors in the development and course of stress-related psychiatric disorders such as major depression. To delineate the role of the corticotropin-releasing hormone receptor type 1 (CRH-R1) in the interactions between corticotropin-releasing hormone and serotonergic neurotransmission,
in vivo microdialysis was performed in CRH-R1-deficient mice under basal (home cage) and stress (forced swimming) conditions. Hippocampal dialysates were used to measure extracellular levels of serotonin and its metabolite 5-hydroxyindoleacetic acid, and free corticosterone levels to monitor the status of the hypothalamic–pituitary–adrenocortical axis. Moreover, behavioural activity was assessed by visual observation and a scoring paradigm.
Both wild-type and heterozygous mutant mice showed a clear diurnal rhythm in free corticosterone. Free corticosterone concentrations were, however, lower in heterozygous mutant mice than in wild-type animals and undetectable in homozygous CRH-R1-deficient mice. Homozygous CRH-R1-deficient mice showed enhanced hippocampal levels of 5-hydroxyindoleacetic acid but not of serotonin during the light and the dark phase of the diurnal cycle, which may point to an enhanced synthesis of serotonin in the raphe-hippocampal system. Moreover, the mutation resulted in higher behavioural activity in the home cage during the light but not during the dark period. Forced swimming caused a rise in hippocampal serotonin followed by a further increase after the end of the stress paradigm in all genotypes. Homozygous and heterozygous mutant mice showed, however, a significantly amplified serotonin response to the forced swimming as compared to wild-type control animals.
We conclude that CRH-R1-deficiency results in reduced hypothalamic–pituitary–adrenocortical axis activity, in enhanced synthesis of serotonin during basal conditions, and in an augmented response in extracellular levels of serotonin to stress. These data provide further evidence for the intricate relationship between corticotropin-releasing hormone and serotonin and the important role of the CRH-R1 herein.
PIASx belongs to the PIAS protein family, the members of which modulate activities of several transcription factors and act as E3 ligases in the sumoylation pathway. The PIASx gene is highly ...expressed in testis, suggesting a role in spermatogenesis. To investigate the function of PIASx in vivo, we have disrupted the PIASx gene in mice. Interestingly, the knockout mice were viable and fertile. Despite the normal fertility, the testis weight of the mutant animals was reduced and their number of apoptotic testicular cells was increased. Also, the sperm count of mutant mice tended to be reduced, but the quality of their sperm cells was normal. No significant changes were observed in the serum levels of LH and FSH or in the intratesticular testosterone concentration between the knockout animals and their wild-type littermates. Compensatory increases in other PIAS protein mRNAs were not observed in the knockout mice. These results imply that PIASx is required quantitatively rather than qualitatively for normal spermatogenesis.
Dopamine-producing neurons in the mammalian midbrain have received considerable attention in recent years because of their involvement in diverse neurological and psychiatric human disorders such as ...Parkinson's Disease (PD), schizophrenia and addiction. Although the underlying pathogenic mechanisms of these disorders are far from being understood, it is meanwhile accepted that a combination of genetic predisposition and environmental factors lead to the disease state. More recent evidence also suggests that both neurological and psychiatric disorders result from early disturbances affecting the normal development of the mesencephalic dopaminergic (mesDA) neurons. Understanding the cues directing the generation of the different mesDA cell groups, the establishment of their proper connections within the brain and their maintenance in the adult are therefore also of great clinical interest. Rodents, and in particular the mouse, have served as the classical "surrogate" organism for these studies based on their phylogenetic relationship to humans, their relatively well characterized mesDA system on both the anatomical and physiological levels, and especially on the propensity of the mouse to genetic manipulation enabling the dissection of genetic pathways underlying the proper generation and maintenance of the mesDA system in this species. In the present review, we will summarize recent findings in the overall context of murine mesDA neuron development.
The cerebellum is an ideal system to study pattern formation in the central nervous system because of its simple cytoarchitecture and regular organization of folds and neural circuitry. Engrailed-2 ...(En-2) is expressed in a spatially restricted broad band around the mesencephalic-metencephalic junction, a region from which the cerebellum is derived. Mice homozygous for a targeted deletion of the En-2 homeobox, En-2hd, previously have been shown to have an altered adult cerebellar foliation pattern. To address whether the En-2hd allele was hypomorphic, we generated a putative null mutation that makes an N-terminal deletion (ntd). Mice homozygous for this new mutation, En-2ntd, display an identical cerebellar patterning defect, suggesting that both alleles represent null alleles. We also examined the developmental profile of En-2 homozygous mutant cerebellar foliation. This revealed a complex phenotype of general developmental delay and abnormal formation of specific fissures with the most severe morphological disruptions being limited to the posterior region of the cerebellum. The expression of two transgenes, which express lacZ in lobe-specific patterns in the cerebellum, also was found to be altered in En-2 homozygotes, suggesting possible lobe transformations. Finally, during embryogenesis there was a clear delay in fusion of the cerebellar rudiments at the midline by 15.5 d.p.c. This and the expression pattern of En-2 suggests that although cerebellar foliation is largely a postnatal process, the patterning of the cerebellum may begin during embryogenesis and that En-2 plays a critical role in this early process.
Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific ...nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs
, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref.
) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca
handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.
Abstract Corticotropin releasing hormone (CRH) is the central modulator of the mammalian hypothalamic–pituitary–adrenal (HPA) axis. In addition, CRH affects other processes in the brain including ...learning, memory, and synaptic plasticity. Moreover, CRH has been shown to play a role in nerve cell survival under apoptotic conditions and to serve as an endogenous neuroprotectant in vitro . Employing mice overexpressing murine CRH in the CNS, we observed a differential response of CRH-overexpressing mice (CRH-COEhom -Nes) to acute excitotoxic stress induced by kainate compared with controls (CRH-COEcon -Nes). Interestingly, CRH-overexpression reduced the duration of epileptic seizures and prevented kainate-induced neurodegeneration and neuroinflammation in the hippocampus. Our findings highlight a neuroprotective action of CRH in vivo . This neuroprotective effect was accompanied by increased levels of brain-derived neurotrophic factor (BDNF) in CRH-COEhom -Nes mice, suggesting a potential role for BDNF in mediating CRH-induced neuroprotective actions against acute excitotoxicity in vivo.
PDF
