NIBB Center for Transgenic Animals and Plants was established in April 1998 to support researches using transgenic and gene targeting techniques in NIBB. The center building for transgenic animals has been completed in the end of 2003 (Figure 1). The building has a floor space of 2,300m², in which we can breed, develop, store and analyze transgenic, gene targeting and mutant mice under condition of “specific pathogen free”. It is also equipped with breeding areas for transgenic aquatic animals, birds and insects.
The expected activities of the Center are as follows:
1. Provision of information, materials, experimental techniques and breeding space of trausgenic animals and plants to researchers.
2. Equipment of various kinds of instruments to analyze mutant, trangenic, and gene-targeted animals and plants.
3. Development of novel techniques related to transgenic and gene targeting technology.

II-I. Academic activity (Watanabe Lab, Physiological role of Nax sodium channel)

We are studying the functional role of Nax (also called Nav2 or NaG) sodium channel in collaboration with Division of Molecular Neurobiology. Nax sodium channel was long classified as a subfamily of voltage-gated sodium channels (NaChs) that serve to generate action potentials in electrically excitable cells such as neuronal and muscle cells. Comparing with the other NaChs, however, Nax has unique amino acid sequences in the regions, which are known to be involved in ion selectivity and voltage-dependent activation and inactivation, suggesting that it must have specific functional properties.

To clarify the functional role of Nax, Nax-deficient mice were generated by gene-targeting technique and the physiological phenotypes have been examined. Behavioral studies suggested that the Nax channel plays an important role in the central sensing of body-fluid sodium level and regulation of salt intake behavior. Nax-deficient mice ingest hypertonic sodium chloride solution in excess in comparison with wild type-mice. LacZ reporter gene knocked into Nax-gene locus revealed that Nax gene is expressed in the circumventricular organs (CVOs), which is the specialized central organs involved in sensing of sodium concentration and osmosity in body fluids. Sodium ion imaging and electrophysiological studies using cultured CVO neurons also suggested that Nax is an extracellular sodium-level sensitive sodium channel. Recently, we found that Nax is expressed in non-myelinating Schwann cells and alveolar type II cells in addition to the neurons and ependymal cells in the CVOs. Nax is thus likely to be involved in reception of sodium-level in body fluids at the CVOs and sodium absorption in the visceral nervous system and in the lung (Figure 2).

More recently, we found in collaboration with Prof. Yamamoto’s group of Osaka University that the peripheral nervous system has only subtle effects on the higher preference for sodium chloride as ovserved in the mutant mice. The results suggest that the mutant phenotype is mainly due to the lack of Nax channels in the central nervous system.

We are now trying to construct functional expression systems of Nax sodium channel using various heterologous cell lines. The heterologous expression system will provide us useful information on the channel charactors of Nax.

Figure 1. A new center facility for transgenic animals built at the area E approximately 1 km away from the area A.

II-II. Academic activity (Sasaoka Lab)

(1) Studying the function of dopaminergic transmission

The dopamine receptors are divided into two subgroups, referred to as D1-like (D1, D5) and D2-like (D2, D3 and D4) receptors on the basis of their gene structure and their pharmacological and transductional properties. The dopaminergic system is implicated in the regulation of the several peptide hormones in the pituitary, the modulation of motor activity, the modulation of synaptic plasticity, and the neural development. The dopaminergic system is also implicated in motivated behaviors, several neurological and psychiatric disorders, such as Parkinson’s disease and schizophrenia. Since D1-like and D2-like receptors often work synergistically, it is necessary to delete both D1-like and D2-like receptors in order to understand the role of dopaminergic transmission. We have generated multiple dopamine receptor-deficient mice by combination of single mutant mice, and are studying phenotype of the multiple dopamine receptor mutant mice.

(2) Developing a novel conditional mutagenesis technique in mice

The aim of the study is to overcome the limitations of the conventional mouse molecular genetic approach in the functional analysis of target genes. We substituted one critical amino acid residue of N-methyl-D-aspartate receptor (NMDAR), leading to NMDAR activation. By our technique, we accomplished conditional substitution of the amino acid in mice and our mutant mice exhibited NMDAR activation and a neurological phenotype, similar to that of mouse models for neurological disorders. The development of our mutant mice should contribute to understanding the function of the critical amino acid residue and the mechanism of neurological disorders. Our new technique is vastly applicable to functional analysis of any desired gene and should contribute to studies on the structural and functional relationships of relevant genes.

(3) Studying of the function of the sarcoglycan complex (SGC)

Sarcoglycans (SG) are trans-sarcolemmal glycoproteins, which associate together to form SGC and present in the sarcolemma. SGC, together with dystrophin and the dystroglycan complex comprises the dystrophin complex, which has been considered as the mechanical link between the basement membrane and the intracellular cytoskeleton for protecting the sarcolemma from mechanical stress during muscle contraction. Each of four SG subunits (a-, b-, g- and d-SG) is responsible for four respective forms of SG-deficient muscular dystrophy, sarcoglycanopathy (SGP). All of the SGs and sarcospan are absent in the sarcolemma in any form of SGP, suggesting that the SGC is not assembled, if a single subunit of the SGC is absent.

To understand a physiological role of the SGC, we generated the b-SG-deficient (BSG-/-) and g-SG-deficient (GSG-/-) mice. The dystrophin complex isolated from the skeletal muscles of BSG-/- mice was unstable in the absence of the SGC and sarcospan. This indicates that SGC and sarcospan play an important role in stabilizing the dystrophin axis connecting the basement membrane and the cytoskeleton. The BSG-/- and GSG-/- mice developed progressive muscular dystrophy with notable muscle hypertrophy. We found that the number of muscle fibers increased with age and most of the fibers were regenerating fibers in their hypertrophic muscle. Therefore, muscle hypertrophy is a consequence of excess regenerating process but not due to fibrous and fat tissue replacement, which has been assumed to be present in so-called pseudohypertrophy in human diseased muscle.

Figure 2. A hypothetical scheme for the physiological role of Nax sodium channel. (a) Nax channel is activated by increase of extracellular sodium concentration. The activated Nax may promote the excitability of GABAergic neurons in SFO. The activated GABAergic neurons in SFO will inhibit the salt-intake-promoting neurons. (b) Repetitive action potentials cause extracellular increase of potassium concentration. Nax may contribute to the potassium uptake by Schwann cells. (c) The osmotic gradient between intra-alveolar space and capillary is a major driving force for the removal of water through water channel expressed in ATI cells. Nax may be one of the molecules generating the osmotic gradient.