Transgenic mice have been used for analyses of cis-acting elements which is involved in the tissue-specific and developmental-specific expression, for analyses of physiological function of genes, or for the production of human disease model. This approach is especially successful in the field of Genetics and Immunology. For the past several years we have been studying one of human genetic diseases, Familial amyloidotic polyneuropathy (FAP) . FAP is one type of systemic amyloidosis and is an autosomal dominant disorder characterized by the extracellular deposition of amyloid fibrils and by prominent peripheral and autonomic nerve involvement. Accumulating evidences suggest that the main cause of this disease is the presence of the mutant transthyretin (TTR) gene. However, many questions in relation to the disease process remain to be elucidated. In addition, there is no effective therapy for FAP. To elucidate the pathological process of this disease development and to devise a new method for treatment, we have produced a transgenic mouse model of FAP by introducing the human mutant transthyretin (hMet30) gene. In these transgenic mice, amyloid deposition starts to occur at around 6 months of age and the amount of amyloid deposition increases gradually with aging. Amyloid deposition is observed in many tissues including heart, kidney and thyroid gland, where amyloid deposition is commonly observed in FAP patient. There was a big variation at the age of onset although we used an inbred strain of mouse, C57BL/6, in these experiments and the serum concentrations of hMet30 were about the same in these transgenic mice. The hMet30 gene was found to be expressed from fetal stage. These results suggest that amyloid deposition itself starts to occur late in life and that this amyloid deposition is influenced by environmental factors.
Several years ago we showed that the major histocompatibility complex (MHC) class II gene is identical to the immune response gene by demonstrating that the immune response can be restored by the new expression of class II molecules on immunocompetent cells. Recent evidence suggest that class II molecule is involved in the generation of autoimmune disease, such as insulin dependent diabetes mellitus (IDDM) . NOD (non-obese diabetic) mouse is shown to be a mouse model for human IDDM. Concerning the class II genes, NOD mouse has two characteristic features, the lack of I-E and the presence of unique I-A. In order to examine the role of these class II molecules, we produced NOD transgenic mice by introducing murine class II genes. The incidences of insulitis in I-E and I-A transgenic mice were much lower than that of control NOD mice suggesting that both the lack of I-E and the presence of unique I-A are responsible for the development of insulitis in NOD mouse. Our data also suggest that the amino acid at position 57 of A^β chain is not the only cause for insulitis.
Recent technical advance in gene targeting together with the conventional transgenic tecnnique will be a powerful tool not only for the establishment of a transgenic mouse model for the human disease but also for analyses of disease process of human disease.

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In 1979, we established an analbuminemic rat mutant strain (Nagase Analbuminemia Rat; NAR) from Sprague-Dawley rats. Using NARs, a number of important findings were obtained. This paper is focused on the genetic analysis, mapping of genes, and the structural analysis of albumin gene in NAR.
Analbuminemia in NAR is inherited as an autosomal recessive trait (designated alb) . The albumin gene is mapped tandem to hooded (h), Vitamin D-binding protein (Gc), and Alb loci in VI linkage group of chromosome 14, h-Gc; 15.5 ± 1.0%, h-Alb; 15.8 ± 1.0%, and Gc-Alb; 0.32 ± 0.16%, respectively. The alb gene has a seven-base-pair deletion in HI intron. This mutation blocks albumin mRNA splicing in NAR liver. NAR might be the most useful model for the understanding mechanism of splicing in a high animal species.

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In this report we describe our recent histological and biochemical studies to characterize the pathogenic mechanisms of cerebellar hypoplasia exhibited by homozygous Sprague-Dawley-strain Gunn rats and gene mapping of bilirubin UDP-glucuronosyltransferase (BRGT) .
Gunn rats are a mutant strain with autosomal recessive trait of hyperbilirubinemia. Homozygous Sprague-Dawley-strain Gunn rats shows marked hyperbilirubinemia and the cerebellar development of the rats is damaged. The severely damaged parts in the cerebellum are known to be anterior and middle lobes while posterior (lobules IXc, IXd and X) are less affected. The most effective day of bilirubin on the cerebellar development is centered on postnatal day 7. It is suggested that the preferential deposition of bilirubin in Purkinje cells of anterior and middle lobes may occur in jaundiced Gunn rat infants, by which the cells are damaged eventually leading to degeneration. The genetic defect in Gunn rats is reported to be deficient in both hepatic BRGT and 3-methylcholanthrene-inducible 4-nitrophenol UDP-glucuronosyltransferase (4-NP-GT) . Both BRGT and 4-NPGT are located on the same chromosome. Sato et al. (1990) isolated a 1763-bp cDNA for rat liver BRGT. The BRGT activity detected by autoradiography on a thin layer chromatogram by transfection of the vector carrying the cDNA into COST cells. The cDNA shares an identical 913-bp sequence with that for rat liver 4-NPGT. A-1 frameshift was found in the locus of 4-NPGT of jaundiced Gunn rats. The result suggests that the multiple defects of UDP-glucuronosyltransferase isoenzymes observed in the Gunn rats may be produced by a single-mutated-locus after an alternative splicing of the identical 913-bp region.

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Many spontaneous, chemical-induced, and radiation induced dominant white spot (W) and steel (Sl) mutation have been identified in the mouse. W and Sl mutations have been similar phenotypic effects including deficiencies in pigment cells, germ cells, hematopoietic stem cells, and mast cells. Numerous studies have suggested that W acts within the affected cell while Sl instead exerts its effects in the extracellular environment. Recently findings demonstrating that W encodes the c-kit prot-oncogene, a tyrosine kinase membrane receptor, have suggested that Sl encodes a ligand for c-kit. We studied the expression of c-kit mRNA in various tissues and cell lines of mouse. And then, we have partially purified c-kit ligand from conditioned medium of BALB /3T3 fibrobrasts. We investigated the effect for proliferation and differentiation to c-kit expressing cells, mast cell, Leydig cell by the partially purified ligand.

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It is generally held that one of the recessive genes controlling diabetes in the NOD mouse is linked to the major histocompatibility complex (MHC) . We therefore performed restriction fragment length polymorphism studies of MHC (class I, II, and III) in NOD mice in comparison with those of their nondiabetic sister strains, NON, CTS, and ILI mice which were derived from the same Jcl-ICR mice. When a minimum of four restriction enzymes were used, class II and III genes of NOD mice were indistinguishable from those of CTS and ILI mice but totally different from those of NON mice. While NON mice expressed the E_α gene, NOD, CTS, and ILI mice appeared to carry a deletion in the 5' end of the E_α gene resulting in failure to transcribe the E_α gene. When class I probe was used, CTS mice showed very different band patterns from those of the other ICR-derived mice.
Unique substitution of Asp57 with Ser in the A_β chain is considered to make the A_β gene the MHC-linked susceptibility gene. We therefore analyzed the nucleotide sequences of the A_β second exon in ILI, CTS, and NON mice. The DNA sequence analyses revealed that the A_β second exon sequences in the ILI and CTS mice, but not in the NON mouse, are identical to that of the NOD mouse. Taken together, these data suggest that ILI and CTS mice possess a recessive diabetogenic gene linked to the MHC.
We also examined the difference of V_β usage between the NOD mouse and the ILI mouse spleen cells. No obvious difference, however, was evident.

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Eye lens obsolescence (Elo) is a heritable, autosomal dominant eye disorder in mice. The Elo mutation lies close to the Len-1 locus on mouse chromosome 1. As it was previously speculated that the Elo locus affects the expression level of γ-crystallin, we first compared the amount of lens γ-crystallin between Elo and normal mice using the 2D-gel electrophoresis. The lens extracts from 10-day-old Elo mice revealed the same profiles and expression levels of α- and β-crystallins as those of +/+ mice. The lower γ-crystallin level could be detected as early as the 14-days stage in the lens of the Elo mouse embryo. The concentration of γ2-crystallin mRNA in Elo mice lens was estimated at 1/13 of that of normal lens, indicating that a small amount of transcript production from γ-crystallin genes may cause the low level of γ-crystallin in Elo lens.
To map precisely the mutation locus of Elo on mouse chromosome 1, linkage analysis was achieved using backcross mating between Elo and 129/SvJ (+/+) . Restriction endonuclease Dra I showed distinct RFLP patterns in both y2- and y4-crystallin genes. The backcross offsprings were analyzed with respect to Elo, Idh-1, Cryg-1, and Cryg-4 loci among 223 mice.
Deficiency in a 94-kDa peptide in the non-crystallin fraction from the Elo mouse lens was shown. An antibody was raised against the 94-kDa peptide isolated from normal mouse lens. The 94-kDa peptide was only observed in the lens, and was unique to mouse lens cortex and nucleus fractions, not being present in epithelial cells. The obtained antibody reacted but weakly with the avian CP97 lens peptide. From these results, we concluded that the 94-kDa antigen corresponds to a lens fiber cellspecific cytoskeletal protein of the mouse. The peptide was deficient in the lenses from Elo mice, but it was contained at the normal level in microphthalmic lenses from CTA mice. The fact indicates that the peptide is not lacking in all microphthalmic lens. It is possible that the suppression of expression of this 94-kDa peptide causes the defective elongation in Elo lens fiber cells.

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The classical form of congenital adrenal hyperplasia (CAH) in humans is caused by a deficiency in steroid 21-hydroxylase (21-OHase) . The enzyme 21-OHase plays a key role in adrenal steroidogenesis. Duplicated genes for the enzyme are located in the class III region of the major histocompatibility complex (MHC), HLA. In the mouse, the genes encoding 21-OHase have been mapped to the homologous region of the H-2 complex. We have previously obtained an intra-H-2 recombinant haplotype aw18 from a cross between H-2 congenic strains of B10. A (H-2^a) and B10. MOL-SGR (H-2^wm7), which carries the H-2 complex derived from the Japanese wild mouse. When mice that were heterozygous for aw18 were intercrossed, no mice that were homozygous for the aw18 haplotype were detected among live offspring of more than 15 days of age, which suggested presence of a recessive lethal gene in the H-2^aw18 haplotype. Molecular analysis of the H-2^aw18 chromosome has revealed a deletion in the H-2 class III region encompassing the gene for the complement component C4 and one of two genes for 21-OHase. We now report that newborn aw18 homozygous mice are deficient in 21-OHase activity, and that homozygosity of the aw18 haplotype directly causes death at the early postnatal stage. Morphological changes in the adrenal glands of newborn aw18 homozygotes are also observed. The aw18 recombinant haplotype is expected to serve as a useful animal model for the inherited human disease of CAH.

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Myelin is an important structure for facilitating the conduction of impulses along the axons both in the central nervous system (CNS) and peripheral nervous system. Myelin basic protein (MBP) is a major protein in the CNS myelin. The MBP gene has been cloned and well characterized. Two mutant mice shiverer and myelin deficient (mid) are autosomal recessive mutants that show severe symptoms such as intentional tremor. They have been found to have a mutation in the MBP gene that results in poor myelination in the CNS. It was found that rearrangement within the MBP gene results in low expression of the gene. In shiverer, MBP gene is partially deleted and MBP mRNA is essentially absent. In mld, the content of MBP in the CNS is greatly reduced, but MBP is clearly detected. Molecular genetic study revealed that MBP gene is duplicated tandemly and a large portion of the duplication is inverted upstream of the intact copy. In the present study, to elucidate the mechanism of repression of MBP gene expression, we performed RNase protection studies. And antisense RNA complementary to exons 3 and 7, which correspond to the inverted segment, was detected. This abnormal transcript was shown to localize in nucleus, and to form an RNA: RNA duplex with sense RNA. These findings suggested that inhibition of transport from the nucleus or selective degradation of the duplex is responsible to for the reduced expression of the MBP gene in the mld mutant. The mechanism of gene rearrangement at the mid locus was also characterized. Cosmid clones encompassing whole MBP gene loci from control and mld genomic library were isolated. The recombination points indicated that the duplication and inversion observed in mld occurred due to nonhomologous recombination.

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