Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi.

BACKGROUND: Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. RESULTS: We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. CONCLUSION: Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.

Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation.

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations.

Somatostatin receptor 2 knockout/lacZ knockin mice show impaired motor coordination and reveal sites of somatostatin action within the striatum.

The peptide somatostatin can modulate the functional output of the basal ganglia. The exact sites and mechanisms of this action, however, are poorly understood, and the physiological context in which somatostatin acts is unknown. Somatostatin acts as a neuromodulator via a family of five 7-transmembrane G protein-coupled receptors, SSTR1-5, one of which, SSTR2, is known to be functional in the striatum. We have investigated the role of SSTR2 in basal ganglia function using mice in which Sstr2 has been inactivated and replaced by the lacZ reporter gene. Analysis of Sstr2lacZ expression in the brain by beta-galactosidase histochemistry demonstrated a widespread pattern of expression. By comparison to previously published in situ hybridization and immunohistochemical data, Sstr2lacZ expression was shown to accurately recapitulate that of Sstr2 and thus provided a highly sensitive model to investigate cell-type-specific expression of Sstr2. In the striatum, Sstr2 expression was identified in medium spiny projection neurons restricted to the matrix compartment and in cholinergic interneurons. Sstr2 expression was not detected in any other nuclei of the basal ganglia except for a sparse number of nondopaminergic neurons in the substantia nigra. Microdialysis in the striatum showed Sstr2-null mice were selectively refractory to somatostatin-induced dopamine and glutamate release. In behavioural tests, Sstr2-null mice showed normal levels of locomotor activity and normal coordination in undemanding tasks. However, in beam-walking, a test of fine motor control, Sstr2-null mice were severely impaired. Together these data implicate an important neuromodulatory role for SSTR2 in the striatum.

Mouse respiratory epithelial cells support efficient replication of human rhinovirus.

Human rhinoviruses (HRV) are responsible for the majority of virus infections of the upper respiratory tract. Furthermore, HRV infection is associated with acute exacerbation of asthma and other chronic respiratory diseases of the lower respiratory tract. A small animal model of HRV-induced disease is required for the development of new therapies. However, existing mouse models of HRV infection are difficult to work with and until recently mouse cell lines were thought to be generally non-permissive for HRV replication in vitro. In this report we demonstrate that a virus of the minor receptor group, HRV1B, can infect and replicate in a mouse respiratory epithelial cell line (LA-4) more efficiently than in a mouse fibroblast cell line (L). The major receptor group virus HRV16 requires human intercellular adhesion molecule-1 (ICAM-1) for cell entry and therefore cannot infect LA-4 cells. However, transfection of in vitro-transcribed HRV16 RNA resulted in the replication of viral RNA and production of infectious virus. Expression of a chimeric ICAM-1 molecule, comprising mouse ICAM-1 with extracellular domains 1 and 2 replaced by the equivalent human domains, rendered the otherwise non-permissive mouse respiratory epithelial cell line susceptible to entry and efficient replication of HRV16. These observations suggest that the development of mouse models of respiratory tract infection by major as well as minor group HRV should be pursued.