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Ken-ichi Yamamura

Publications and source records attributed to Ken-ichi Yamamura.

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

Meltrin beta expressed in cardiac neural crest cells is required for ventricular septum formation of the heart.

The heart is divided into four chambers by membranous septa and valves. Although evidence suggests that formation of the membranous septa requires migration of neural crest cells into the developing heart, the functional significance of these neural crest cells in the development of the endocardial cushion, an embryonic tissue that gives rise to the membranous appendages, is largely unknown. Mice defective in the protease region of Meltrin beta/ADAM19 show ventricular septal defects and defects in valve formation. In this study, by expressing Meltrin beta in either endothelial or neural crest cell lineages, we showed that Meltrin beta expressed in neural crest cells but not in endothelial cells was required for formation of the ventricular septum and valves. Although Meltrin beta-deficient neural crest cells migrated into the heart normally, they could not properly fuse the right and left ridges of the cushion tissues in the proximal outflow tract (OT), and this led to defects in the assembly of the OT and AV cushions forming the ventricular septum. These results genetically demonstrated a critical role of cardiac neural crest cells expressing Meltrin beta in triggering fusion of the proximal OT cushions and in formation of the ventricular septum.

ADAM Proteins↗
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

The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse.

Gene trapping is a method of generating murine embryonic stem (ES) cell lines containing insertional mutations in known and novel genes. A number of international groups have used this approach to create sizeable public cell line repositories available to the scientific community for the generation of mutant mouse strains. The major gene trapping groups worldwide have recently joined together to centralize access to all publicly available gene trap lines by developing a user-oriented Website for the International Gene Trap Consortium (IGTC). This collaboration provides an impressive public informatics resource comprising approximately 45 000 well-characterized ES cell lines which currently represent approximately 40% of known mouse genes, all freely available for the creation of knockout mice on a non-collaborative basis. To standardize annotation and provide high confidence data for gene trap lines, a rigorous identification and annotation pipeline has been developed combining genomic localization and transcript alignment of gene trap sequence tags to identify trapped loci. This information is stored in a new bioinformatics database accessible through the IGTC Website interface. The IGTC Website (www.genetrap.org) allows users to browse and search the database for trapped genes, BLAST sequences against gene trap sequence tags, and view trapped genes within biological pathways. In addition, IGTC data have been integrated into major genome browsers and bioinformatics sites to provide users with outside portals for viewing this data. The development of the IGTC Website marks a major advance by providing the research community with the data and tools necessary to effectively use public gene trap resources for the large-scale characterization of mammalian gene function.

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

Immunization in familial amyloidotic polyneuropathy: counteracting deposition by immunization with a Y78F TTR mutant.

The mechanism of amyloid formation in familial amyloidotic polyneuropathy (FAP), a hereditary disorder associated with mutant transthyretin (TTR), is still unknown. It is generally believed that altered conformations exposing cryptic regions are intermediary steps in this mechanism. A TTR mutant--Y78F (transthyretin mutant with phenylalanine replacing tyrosine at position 78)--designed to destabilize the native structure has been shown to expose a cryptic epitope recognized by a monoclonal antibody that reacts only with highly amyloidogenic mutants presenting the amyloid fold or with amyloid fibrils. To test whether TTR deposition in FAP can be counteracted by antibodies for cryptic epitopes, we immunized with TTR Y78F, transgenic mice carrying the most common FAP-associated TTR mutant--V30M (transthyretin mutant with methionine replacing valine at position 30)--at selected ages that present normally with either nonfibrillar or TTR amyloid deposition. Compared to age-matched control nonimmunized mice, Y78F-immunized mice had a significant reduction in TTR deposition usually found in this strain, in particular in stomach and intestine; by contrast, animals immunized with V30M did not show differences in deposition in comparison with nonimmunized mice. Immunohistochemical analyses of tissues revealed that immunization with Y78F lead to infiltration by lymphocytes and macrophages at common deposition sites, but not in tissues such as liver, choroid plexus, and Langerhans islets, in which TTR is produced. These results suggest that Y78F induced production of an antibody that reacts specifically with deposits and leads to an immune response effective in removing/preventing TTR deposition. Therefore, TTR immunization with selected TTR mutants has potential application in immune therapy for FAP.

Amyloid Neuropathies, Familial↗
PubMed2006

Abnormal migration and distribution of neural crest cells in Pax6 heterozygous mutant eye, a model for human eye diseases.

PAX6/Pax6 gene encodes a transcription factor that is crucially required for eye development. Pax6 heterozygous mutant mouse (Pax6(Sey/+)) shows various ocular defects, especially in the anterior segment. It has been well known that the induction of the lens and development of the cornea and retina are dependent on PAX6/Pax6 in a cell-autonomous fashion, although the influence of PAX6/Pax6 on the other tissues derived from the ocular mesenchyme is largely unknown. Using transgenic mouse lines in which neural crest cells are genetically marked by LacZ or EGFP, we revealed the extensive contribution of neural crest derived cells (NCDCs) to the ocular tissues. Furthermore, various eye defects in Pax6(Sey/+) mouse were accompanied by abnormal distribution of NCDCs from early developmental stages to the adult. In Pax6(Sey/+) mouse mice, neural crest cells abnormally migrated into the developing eye in a cell nonautonomous manner at early embryonic stages. These results indicate that normal distribution and integration of NCDCs in ocular tissues depend on a proper dosage of Pax6, and that Pax6(Sey/+) eye anomalies are caused by cell autonomous and nonautonomous defects due to Pax6 haploinsufficiency.

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

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

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

Deposition of transthyretin amyloid is not accelerated by the same amyloid in vivo.

Acceleration of amyloid deposition by administration of amyloid fibrils and transmissibility of disease have been reported in several types of amyloidoses. Families with a variant transthyretin (TTR V30M)-associated familial amyloidotic polyneuropathy (FAP) exhibit genetic anticipation, with TTR V30M-amyloid depositing at an earlier age in successive generations. The molecular bases of anticipation in FAP have remained to be determined. We asked if administration of TTR-amyloid fibrils (ATTR) extracted from the heart of an FAP TTR V30M patient would accelerate ATTR deposition in transgenic mice expressing the human mutant ttr gene responsible for FAP TTR V30M and indeed the administration did accelerate deposition of apolipoprotein A-II-amyloid fibrils (AApoAII), and not A TTR. Our experiments present, for the first time, evidence that the degree of inducibility of ATTR is low relative to AApoAII and we suggest that administration of ATTR may not explain the genetic anticipation which occurs in FAP.

Amyloid↗
PubMed2004

Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus.

Transgenesis using large DNA such as YAC or BAC has extended the range of applications in functional genomics. Here we describe an efficient BAC transgenesis protocol using a simple BAC DNA preparation method adopted from YAC DNA purification methods. This method allowed us to isolate BAC DNA from small scale culture of BAC-containing cells in sufficient quantity and purity for microinjection. More than 40 founders have been produced with linearized BAC DNA prepared by this method, and 85% of them contained intact BAC transgenes. In contrast, when circular BAC DNA was injected, an approximately three-fold reduction of transgene integration rate was observed and fewer intact transgene integrations were obtained. A line of transgenic mice carrying a 170-kb BAC clone generated in this way successfully rescued tail and embryonic lethality phenotypes of the mouse Brachyury (T) mutants, further demonstrating the utility of this method in functional analysis of the mouse genome.

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

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↗