Biomedical subjects

Masatake Araki

Publications and source records attributed to Masatake Araki.

11 recordsLinked to original sources

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

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↗

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

Generation of high-affinity antibody against T cell-dependent antigen in the Ganp gene-transgenic mouse.

Generation of high-affinity Ab is impaired in mice lacking germinal center-associated DNA primase (GANP) in B cells. In this study, we examined the effect of its overexpression in ganp transgenic C57BL/6 mice (Ganp(Tg)). Ganp(Tg) displayed normal phenotype in B cell development, serum Ig levels, and responses against T cell-independent Ag; however, it generated the Ab with much higher affinity against nitrophenyl-chicken gammaglobulin in comparison with C57BL/6. To further examine the affinity increase, we established hybridomas producing high-affinity mAbs and compared their affinities using BIAcore. C57BL/6 generated high-affinity anti-nitrophenyl mAbs (K(D) approximately 2.50 x 10(-7) M) of IgG1/lambda1 and contained the V(H)186.2 region with W33L mutation. Ganp(Tg) generated much higher affinity (K(D) > 1.57 x 10(-9) M) by usage of V(H)186.2 as well as noncanonical V(H)7183 regions. Ganp(Tg) also generated exceptionally high-affinity anti-HIV-1 (V3 peptide) mAbs (K(D) > 9.90 x 10(-11) M) with neutralizing activity. These results demonstrated that GANP is involved in V region alteration generating high-affinity Ab.

Amino Acid Sequence↗

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↗

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

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

Naso-maxillary deformity due to frontonasal expression of human transthyretin gene in transgenic mice.

BACKGROUND: Retinoic acid, a metabolic product of retinol, is essential for craniofacial morphogenesis. Transthyretin (TTR) is a plasma protein delivering retinol to tissues. We produced several transgenic mouse lines using the human mutant TTR (hTTRMet30) gene to establish a mouse model of familial amyloidotic polyneuropathy. One of the lines showed an autosomal dominant inheritance of naso-maxillary deformity termed Nax. RESULTS: The Nax malformation was characterized by a hypoplastic developmental defect of the frontonasal region. Homozygous mice with higher transgene expressions showed more severe phenotypes, but a subline, in which the copy number and expression of the transgene was reduced, showed a normal phenotype, indicating that the hTTRMet30 expression caused the malformation. Nax mice began to express the hTTRMet30 gene in the nasal placode from embryonic day 10.5 (E10.5), which was 2 days earlier than in the other transgenic lines with a normal phenotype. Excessive cell death was observed in the nasal placode of the E10.5 Nax embryos. In addition, the forced expression of hTTRMet30 in the nasal placode of transgenic mice resulted in similar phenotypes. CONCLUSION: The expression of the hTTRMet30 gene in the nasal placode at E10.5 induced apoptotic cell death, leading to hypoplastic deformity in the frontonasal region.

Animals↗

PubMed2002

Region-specific gastrointestinal Hox code during murine embryonal gut development.

Hox genes encode transcription factors, and they are involved in the specification of each body part along the anteroposterior (AP) body axis during embryogenesis. To clarify AP pattern formation of the digestive tract, the expression patterns of Hox genes belonging to paralogous groups 4 and 5, and parts of groups 6 and 7, were systematically examined by whole-mount and section in situ hybridization. The Hox gene expression pattern of paralogous groups 4-9 in the developing gut at 12.5 days post-coitum was fully examined. All HoxA and HoxB genes in paralogous groups 4-8 were expressed in the stomach, in contrast to the HoxC and HoxD genes. In the midgut region, all Hox cluster genes showed colinear expression within each cluster, yielding the Hox code; the more 3' located genes were expressed more rostrally and the 5' group genes more caudally. The colinear expression of HoxA and HoxB cluster genes started from the duodenum, that of HoxC cluster genes started from the jejunum, and HoxD cluster genes were expressed in the caudal part of the midgut, ileum and cecum. In the hindgut region, HoxD cluster genes and Abd-B family genes were expressed. Thus, a different Hox code seems to exist in each subdomain of developing gut (foregut, midgut and hindgut). The visceral mesoderm restricted expression also suggested that the Hox code primarily functions in mesenchymal specification, and then leads to the regional differentiation of gut subdomains as the result of epithelial-mesenchymal interactions.

Animals↗

PubMed2002

Suppression of experimental lupus nephritis by aberrant expression of the soluble E-selectin gene.

Circulating leukocytes, particularly neutrophils and monocytes, are important effector cells in the induction of many forms of glomerulonephritis. Adhesion molecules, especially selectins, are also thought to be critical for the development of this disease. We examined the possible suppressive effect of soluble E-selectin on the development of experimental lupus nephritis induced by the injection of a hybridoma clone (2B11.3) derived from an MRL/MpJ-lpr/lpr lupus mouse. This clone produces IgG3 antibodies that induce severe proliferative glomerulonephritis resembling lupus nephritis when injected into normal mice. Transgenic mice with a soluble E-selectin gene were injected intraperitoneally with the hybridoma cells and histopathologically examined on day 15. As a result, the development of glomerulonephritis was significantly suppressed. This suppression was characterized by fewer inflammatory cell infiltrates, compared with non-transgenic litter mates, despite the fact that there were no remarkable differences in immunoglobulin deposits or expression of E-selectin between the two groups. These findings suggest that by controlling inflammatory cell infiltration, soluble E-selectin plays a preventative role in the development of a particular type of lupus nephritis.

Animals↗