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Kimi Araki

Publications and source records attributed to Kimi Araki.

At least 19 recordsLinked to original sources
PubMed2006

LGR4 regulates the postnatal development and integrity of male reproductive tracts in mice.

The roles of the leucine-rich repeat domain containing G protein-coupled receptor (GPCR) 4 (Lgr4), which is one of the orphan GPCRs, were analyzed with the Lgr4 hypomorphic mutant mouse line (Lgr4(Gt)). This homozygous mutant had only one-tenth the normal transcription level; furthermore, 60% of them survived to adulthood. The homozygous male was infertile, showing morphologic abnormalities in both the testes and the epididymides. In the testes, luminal swelling, loss of germinal epithelium in the seminiferous tubules, and rete testis dilation were observed. Cauda epididymidis sperm were immotile. Rete testis dilation was due to a water reabsorption failure caused by a decreased expression of an estrogen receptor (ESR1) and SLC9A3 in the efferent ducts. Although we found differential regulation of ESR1 expression in the efferent ducts and the epididymis, the role of ESR1 in the epididymis remains unclear. The epididymis contained short and dilated tubules and completely lacked its initial segment. In the caput region, we observed multilamination and distortion of the basement membranes (BMs) with an accumulation of laminin. Rupture of swollen epididymal ducts was observed, leading to an invasion of macrophages into the lumen. Male infertility was probably due to the combination of a developmental defect of the epididymis and the rupture of the epithelium resulting in the immotile spermatozoa. These results indicate that Lgr4 has pivotal roles to play in the regulation of ESR1 expression, the control of duct elongation through BM remodeling, and the regional differentiation of the caput epididymidis.

Animals↗
PubMed2006

Negative selection with the Diphtheria toxin A fragment gene improves frequency of Cre-mediated cassette exchange in ES cells.

The Cre-lox system is an important tool for genetic manipulation in embryonic stem cells. We previously reported that the cassette exchange strategy using the mutant lox66/71 and lox2272 combination showed high recombination efficiency and stability. However, the efficiency was strongly affected by the position of chromosomal target lox sites. To enrich successful cassette exchange events, even in clones showing lower recombination efficiency, we have improved exchange vector. The Diphtheria toxin A fragment gene was placed in the un-exchanged region for negative selection and the puromycin N-acetyltransferase gene, instead of the neomycin phosphotransferase gene, was used for positive selection. By reducing random integration, the frequency of successful cassette exchange increased up to 2-4 fold. Furthermore, by adding the third lox site to induce intrarmolecular recombination, the recombination efficiency of cassette exchange itself was improved, and the frequency increased to maximum 5 fold, in which the percentage of exchanged clones reached to 50-70%. This strategy should be useful for other recombinase-mediated cassette exchanges.

Animals↗
PubMed2006

Smooth muscle-specific dystrophin expression improves aberrant vasoregulation in mdx mice.

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Therapeutic options for DMD are limited because the pathogenetic mechanism by which dystrophin deficiency produces the clinical phenotype remains obscure. Recent reports of abnormal alpha-adrenergic vasoregulation in the exercising muscles of DMD patients and in the mdx mouse, an animal model of DMD, prompted us to hypothesize that the dystrophin-deficient smooth muscle contributes to the vascular and dystrophic phenotypes of DMD. To test this, we generated transgenic mdx mice that express dystrophin only in smooth muscle (SMTg/mdx). We found that alpha-adrenergic vasoconstriction was markedly attenuated in the contracting hindlimbs of C57BL/10 wild-type mice, an effect that was mediated by nitric oxide (NO) and was severely impaired in the mdx mice. SMTg/mdx mice showed an intermediate phenotype, with partial restoration of the NO-dependent modulation of alpha-adrenergic vasoconstriction in active muscle. In addition, the elevated serum creatine kinase levels observed in mdx mice were significantly reduced in SMTg/mdx mice. This is the first report of a functional role of dystrophin in vascular smooth muscle.

Animals↗
PubMed2006

Impaired expression of importin/karyopherin beta1 leads to post-implantation lethality.

Importin beta1 (Impbeta)/karyopherin beta1 (Kpnb1) mediates the nuclear import of a large variety of substrates. This study aimed to investigate the requirement for the Kpnb1 gene in mouse development, using a gene trap line, B6-CB-Ayu8108(GtgeoIMEG) (Ayu8108(geo)), in which the trap vector was inserted into the promoter region of the Kpnb1 gene, but in reverse orientation of the Kpnb1 gene. Ayu8108(geo/geo) homozygous embryos could develop to the blastocyst stage, but died before embryonic day 5.5, and expression of the Kpnb1 gene in homozygous blastocysts was undetectable. We also replaced the betageo gene with Impbeta cDNA through Cre-mediated recombination to rescue Impbeta expression. Homozygous mice for the rescued allele Ayu8108(Impbeta/Impbeta) were born and developed normally. These results demonstrated that the cause of post-implantation lethality of Ayu8108(geo/geo) homozygous embryos was impaired expression of the Kpnb1 gene, indicating indispensable roles of Impbeta1 in early development of mice.

Animals↗
PubMed2006

Cloning and expression analysis of a murine novel gene, Ayu17-449.

We used gene trapping vector PU8 to search some interesting genes which play important roles in mouse development from murine ES cells. One positive ES colony termed Ayu17-449 was trapped. Its partial cDNA was obtained by using 5' RACE method. It is homologous to a 5,523 bp cDNA fragment (GI: 20879412) in EST database. Further analysis of the 5,523 bp cDNA sequence in Celera mouse gene database showed that it overlaps two genes. We designed serials of DNA primers according to the mRNAs of these two genes for RT-PCR and Northern blotting analysis, and identified a novel RNA about 9 kb (we named it as Ayu17-449) encoding 1,920 aa. This gene is expressed highly in the brain, kidney, heart, lung, muscle and stomach. The expressed protein contains a Granin motif on its N-terminus, showing that this gene may be involved in hormone secretion.

Animals↗
PubMed2006

[Generation of Ayu17-449 gene knockout mice by gene trapping].

We report the generation of Ayu17-449 gene knockout mice in an effort to facilitate studies of its function during mouse development, A special gene trapping vector was transfected into mouse ES cells. The clone with the trapped gene trap was identified by 5' RACE and Southern blot analysis. Ayu17-449 trapped mice were then generated by using this ES clone. In addition, the expression of Ayu17-449 in this mutant line was analyzed with Northern blot. Results showed that in these mutant mice, the trapping vector was located upstream of the Ayu17-449 initiation site, and as a result, the transcription of Ayu17-449 gene was inhibited. Ayu17-449 trapped mice are a useful tool for the analysis of Ayu17-449 gene function in mouse development.

Animals↗
PubMed2005

A novel murine gene, Sickle tail, linked to the Danforth's short tail locus, is required for normal development of the intervertebral disc.

We established the mutant mouse line, B6;CB-SktGtAyu8021IMEG (SktGt), through gene-trap mutagenesis in embryonic stem cells. The novel gene identified, called Sickle tail (Skt), is composed of 19 exons and encodes a protein of 1352 amino acids. Expression of a reporter gene was detected in the notochord during embryogenesis and in the nucleus pulposus of mice. Compression of some of the nuclei pulposi in the intervertebral discs (IVDs) appeared at embryonic day (E) 17.5, resulting in a kinky-tail phenotype showing defects in the nucleus pulposus and annulus fibrosus of IVDs in SktGt/Gt mice. These phenotypes were different from those in Danforth's short tail (Sd) mice in which the nucleus pulposus was totally absent and replaced by peripheral fibers similar to those seen in the annulus fibrosus in all IVDs. The Skt gene maps to the proximal part of mouse chromosome 2, near the Sd locus. The genetic distance between them was 0.95 cM. The number of vertebrae in both [Sd +/+ SktGt] and [Sd SktGt/+ +] compound heterozygotes was less than that of Sd heterozygotes. Furthermore, the enhancer trap locus Etl4lacZ, which was previously reported to be an allele of Sd, was located in the third intron of the Skt gene.

Amino Acid Sequence↗
PubMed2005

Microarray analysis of the genes induced by tetracycline-regulated expression of NDRF/NeuroD2 in P19 cells.

NeuroD-related factor (NDRF)/NeuroD2 is a basic helix-loop-helix (bHLH) protein that plays important roles in neuronal development. To elucidate the NDRF transcription network, we used mouse cDNA microarray analysis combined with a tetracycline-regulatable expression system in P19 embryonal carcinoma cells. Five genes were identified to be up-regulated in the presence of NDRF protein. RNA hybridization analysis confirmed that brain-lipid-binding protein (BLBP) and inhibitor of differentiation 1 (Id1) genes were among the five genes that were rapidly and significantly up-regulated after induction of NDRF. When a dominant negative form of NDRF protein was expressed during retinoic acid-induced neuronal differentiation of P19 cells, the BLBP gene, but not the Id1 gene, was potently repressed. Immunohistochemical analysis revealed that both NDRF and Id1 immunoreactivities were observed in some granule cells of the cerebellum in the postnatal period. These results suggest that NDRF or its related bHLH proteins may act upstream of these genes in a subset of developing neurons.

Animals↗
PubMed2005

Increase of smooth muscle cell migration and of intimal hyperplasia in mice lacking the alpha/beta hydrolase domain containing 2 gene.

Multiple steps, including the migration of vascular smooth muscle cells (SMCs), are involved in the pathogenesis of atherosclerosis. To discover genes which are involved in these steps, we screened mutant mouse lines established by the exchangeable gene trap method utilizing X-gal staining during their embryonic development. One of these lines showed strong reporter gene expression in the vitelline vessels of yolk sacs at embryonic day (E) 12.5. The trap vector was inserted into the fifth intron of alpha/beta hydrolase domain containing 2 (Abhd2) gene which was shown to be expressed in vascular and non-vascular SMCs of adult mice. Although homozygous mutant mice were apparently normal, enhanced SMC migration in the explants SMCs culture and marked intimal hyperplasia after cuff placement were observed in homozygous mice in comparison with wild-type mice. Our results show that Abhd2 is involved in SMC migration and neointimal thickening on vascular SMCs.

Animals↗
PubMed2005

Autophagic cell death of pancreatic acinar cells in serine protease inhibitor Kazal type 3-deficient mice.

BACKGROUND & AIMS: Serine protease inhibitor Kazal type 1 (SPINK1), which is structurally similar to epidermal growth factor, is thought to inhibit trypsin activity and to prevent pancreatitis. Point mutations in the SPINK1 gene seem to predispose humans to pancreatitis; however, the clinical significance of SPINK1 mutations remains controversial. This study aimed to elucidate the role of SPINK1. METHODS: We generated Spink3-deficient (Spink3(-/-)) mice by gene targeting in mouse embryonic stem cells. Embryonic and neonatal pancreases were analyzed morphologically and molecularly. Specific probes were used to show the typical autophagy that occurs during acinar cell death. RESULTS: In Spink3(-/-) mice, the pancreas developed normally up to 15.5 days after coitus. However, autophagic degeneration of acinar cells, but not ductal or islet cells, started from day 16.5 after coitus. Rapid onset of cell death occurred in the pancreas and duodenum within a few days after birth and resulted in death by 14.5 days after birth. There was limited inflammatory cell infiltration and no sign of apoptosis. At 7.5 days after birth, residual ductlike cells in the tubular complexes strongly expressed pancreatic duodenal homeodomain-containing protein 1, a marker of pancreatic stem cells, without any sign of acinar cell regeneration. CONCLUSIONS: The progressive disappearance of acinar cells in Spink3(-/-) mice was due to autophagic cell death and impaired regeneration. Thus, Spink3 has essential roles in the maintenance of integrity and regeneration of acinar cells.

Animals↗
PubMed2005

Characterization of an exchangeable gene trap using pU-17 carrying a stop codon-beta geo cassette.

We have developed a new exchangeable gene trap vector, pU-17, carrying the intron-lox71-splicing acceptor (SA)-beta geo-loxP-pA-lox2272-pSP73-lox511. The SA contains three stop codons in-frame with the ATG of beta galactosidase/neomycin-resistance fusion gene (beta geo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre-mutant lox system, we successfully replaced the beta geo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp-deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the beta geo gene. Thus, using this pU-17 trap vector, we can initially carry out random mutagenesis, and then convert it to a gain-of-function mutation by replacing the beta geo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre-mediated recombination.

Animals↗
PubMed2004

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos.

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.

Animals↗
PubMed2003

Myopathy phenotype in transgenic mice expressing mutated PABPN1 as a model of oculopharyngeal muscular dystrophy.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized clinically by progressive ptosis, dysphagia and limb weakness, and by unique intranuclear inclusions in the skeletal muscle fibers. The disease is caused by the expansion of a 10-alanine stretch to 12-17 alanine residues in the poly(A)-binding protein, nuclear 1 (PABPN1; PABP2). While PABPN1 is a major component of the inclusions in OPMD, the exact cause of the disease is unknown. To elucidate the molecular mechanism and to construct a useful model for therapeutic trials, we have generated transgenic mice expressing the hPABPN1. Transgenic mice lines expressing a normal hPABPN1 with 10-alanine stretch did not reveal myopathic changes, whereas lines expressing high levels of expanded hPABPN1 with a 13-alanine stretch showed an apparent myopathy phenotype, especially in old age. Pathological studies in the latter mice disclosed intranuclear inclusions consisting of aggregated mutant hPABPN1 product. Furthermore, some TUNEL positive nuclei were shown around degenerating fibers and a cluster of it in the lesion in necrotic muscle fibers. Interestingly, the degree of myopathic changes was more prominent in the eyelid and pharyngeal muscles. Further, muscle weakness in the limbs was apparent as shown by the fatigability test. Nuclear inclusions seemed to develop gradually with aging, at least after 1 week of age, in model mouse muscles. We established the first transgenic mouse model of OPMD by expressing mutated PABPN1, and our model mice appear to have more dramatic alternations in myofiber viability.

Age Factors↗
PubMed2002

Generation and genetic modification of dendritic cells derived from mouse embryonic stem cells.

We developed a method to generate dendritic cells (DCs) from mouse embryonic stem (ES) cells. We cultured ES cells for 10 days on feeder cell layers of OP9, in the presence of granulocyte-macrophage colony-stimulating factor in the latter 5 days. The resultant ES cell-derived cells were transferred to bacteriologic Petri dishes without feeder cells and further cultured. In about 7 days, irregularly shaped floating cells with protrusions appeared and these expressed major histocompatibility complex class II, CD11c, CD80, and CD86, with the capacity to stimulate primary mixed lymphocyte reaction (MLR) and to process and present protein antigen to T cells. We designated them ES-DCs (ES cell-derived dendritic cells), and the functions of ES-DCs were comparable with those of DCs generated from bone marrow cells. Upon transfer to new dishes and stimulation with interleukin-4 plus tumor necrosis factor alpha, combined with anti-CD40 monoclonal antibody or lipopolysaccharide, ES-DCs completely became mature DCs, characterized by a typical morphology and higher capacity to stimulate MLR. Using an expression vector containing the internal ribosomal entry site-puromycin N-acetyltransferase gene or a Cre-lox-mediated exchangeable gene-trap system, we could efficiently generate ES cell transfectants expressing the products of introduced genes after their differentiation to DCs. ES-DCs expressing invariant chain fused to a pigeon cytochrome C epitope presented the epitope efficiently in the context of E(k). We primed ovalbumin (OVA)-specific cytotoxic T lymphocytes in vivo by injecting mice with ES-DCs expressing OVA, thus demonstrating immunization with ES-DCs genetically engineered to express antigenic protein. The methods may be applicable to immunomodulation therapy and gene-trap investigations of DCs.

Animals↗
PubMed2002

A transgenic mouse model of autoimmune glomerulonephritis and necrotizing arteritis associated with cryoglobulinemia.

Mice implanted with hybridoma secreting 6-19 IgG3 anti-IgG2a rheumatoid factor (RF) with cryoglobulin activity develop acute glomerulonephritis and cutaneous leukocytoclastic vasculitis. As the RF activity is implicated in the skin, but not glomerular lesions, it is still unclear whether the renal pathogenicity is determined by 6-19 H chains alone or their combination with L chains. To address this question, we have generated transgenic mice expressing only the H chain gene or both H and L chain genes of the 6-19 IgG3 anti-IgG2a RF and determined the development of glomerular and vascular lesions. H-single and H/L-double transgenic mice displayed comparable high amounts of IgG3 cryoglobulins, but only H/L-double transgenic mice having 10-fold higher levels of IgG3 anti-IgG2a RF progressively developed chronic, lethal glomerulonephritis. The severe glomerular lesions observed at 8-10 mo of age were very heterogeneous (membranoproliferative changes, crescents, and sclerosis); in addition, one-third of them had necrotizing arteritis in the kidneys and skeletal muscles. These renal and vascular changes were very different from those observed in the acute cryoglobulinemia, characterized by mainly "wire-loop" glomerular lesions and a cutaneous leukocytoclastic form of vasculitis. Thus, our data demonstrate the importance of a unique combination of the H and L chains for the expression of the pathogenic activity of IgG3 cryoglobulins and that a single autoantibody is able to induce different types of glomerular and vascular complications, depending on its production levels and kinetics.

Animals↗
PubMed2002

Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites.

The Cre-lox system is an important tool for genetic manipulation. To promote integrative reactions, two strategies using mutant lox sites have been developed. One is the left element/right element (LE/RE)-mutant strategy and the other is the cassette exchange strategy using heterospecific lox sites such as lox511 or lox2272. We compared the recombination efficiencies using these mutant lox sites in embryonic stem (ES) cells, and found that the combination of the LE/RE mutant and lox2272 showed high recombination efficiency and stability of the recombined structure. Taking advantage of this stability, we successfully integrated the cre gene into the mutant lox sites by Cre-mediated recombination. Germ line chimeric mice were produced from the cre-integrated ES cell clones, and Cre-expressing mouse lines were established. The inserted cre gene was stably maintained through the generations. This cre knock-in system using the LE/RE-lox2272 combination should be useful for the production of various cre mice for gene targeting or gene trapping.

Animals↗
PubMed2002

Regulation of vasculogenesis and angiogenesis by EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells.

Although the cellular and molecular mechanisms governing angiogenesis are only beginning to be understood, signaling through endothelial-restricted receptors, particularly receptor tyrosine kinases, has been shown to play a pivotal role in these events. Recent reports show that EphB receptor tyrosine kinases and their transmembrane-type ephrin-B2 ligands play essential roles in the embryonic vasculature. These studies suggest that cell-to-cell repellent effects due to bidirectional EphB/ephrin-B2 signaling may be crucial for vascular development, similar to the mechanism described for neuronal development. To test this hypothesis, we disrupted the precise expression pattern of EphB/ephrin-B2 in vivo by generating transgenic (CAGp-ephrin-B2 Tg) mice that express ephrin-B2 under the control of a ubiquitous and constitutive promoter, CMV enhancer-beta-actin promoter-beta-globin splicing acceptor (CAG). These mice displayed an abnormal segmental arrangement of intersomitic vessels, while such anomalies were not observed in Tie-2p-ephrin-B2 Tg mice in which ephrin-B2 was overexpressed in only vascular endothelial cells (ECs). This finding suggests that non-ECs expressing ephrin-B2 alter the migration of ECs expressing EphB receptors into the intersomitic region where ephrin-B2 expression is normally absent. CAGp-ephrin-B2 Tg mice show sudden death at neonatal stages from aortic dissecting aneurysms due to defective recruitment of vascular smooth muscle cells to the ascending aorta. EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells plays an essential role in vasculogenesis, angiogenesis, and vessel maturation.

Actins↗