Search PubMed⌕ Search

Biomedical subjects

M Obinata

Publications and source records attributed to M Obinata.

169 records · Page 10Linked to original sources
PubMedDate not supplied

Structure of thymidine kinase gene introduced into mouse Ltk- cells by a new injection method.

Pricking, a new injection method developed by Yamamoto et al. (1981), can be used to introduce DNA into cultured cells with high efficiency. Closed circular plasmid DNA containing the cloned HSV-TK gene (pTK-1) was introduced by this method and the structure of DNA in stable transformants was examined. In most clones, the introduced DNA was integrated into the mouse genome in a tandemly repeated form. The possibility of multiple integration via mouse middle repetitive sequences was also examined using the chimeric plasmid with TK genes and middle repetitive sequences (pMRTK-1). Digestion with restriction enzymes showed that the middle repetitive sequence used in this experiment had no effect on the efficiency of transformation, suggesting that this sequence is unable to mediate homologous recombination with mouse genomes.

Animals↗
PubMedDate not supplied

Production of human alpha-interferon in silkworm using a baculovirus vector.

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.

Amino Acid Sequence↗
PubMedDate not supplied

Establishment of Leydig cell line, TTE1, from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

A Leydig cell line, TTE1, has been established from transgenic mice harboring a temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene. The cells grew at a permissive temperature (33 degrees C), but growth was markedly prevented at a nonpermissive temperature (39 degrees C). T-antigen was expressed in the nuclei at 33 degrees C but disappeared at 39 degrees C, indicating that the cells show a temperature-sensitive growth phenotype reflected by the tsSV40 large T-antigen. TTE1 cells did not show any colony-forming activity in soft agar and form tumors in subcutaneous tissue in nude mice, indicating that the cells were not transformed. Alkaline phosphatase and 3beta-hydroxysteroid dehydrogenase (HSD) activities or expression of cytokeratin and vimentin were observed. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that TTE1 cells expressed mRNAs encoding 17beta-HSD types 1 and 3, and inhibin-alpha. The cells with unique characteristics, therefore, should serve useful model study the function of Leydig cell.

17-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Oncostatin m regulates mesenchymal cell differentiation and enhances hematopoietic supportive activity of bone marrow stromal cell lines.

Bone marrow stromal cell lines (TBR cell lines) established from temperature-sensitive Simian Virus 40 T-antigen gene transgenic mice exhibited myogenic, osteogenic, and adipogenic differentiation. The effect of oncostatin M (OSM) on such mesenchymal cell differentiation of marrow stromal cell lines was examined. One of those stromal cell lines, TBRB, differentiated into skeletal muscle, and its differentiation was stimulated by OSM, whereas differentiation of TBR10-1 into smooth muscle was inhibited by OSM. TBR31-2 is a bipotent progenitor for adipocytes and osteoblasts, and OSM stimulated osteogenic differentiation while inhibiting adipogenic differentiation. On the other hand, TBR cell lines exhibited various potentials for supporting hematopoiesis in culture. When hematopoietic progenitor cells were cocultured with OSM-stimulated stromal cell lines, TBR10-1 and TBR31-2 exhibited enhanced hematopoietic supportive activity. As responsible molecules for stromal cell dependent hematopoiesis, expression of stem cell factor (SCF) (a ligand of c-Kit), vascular cell adhesion molecule (VCAM-1) (a ligand of VLA-4), and secretion of interleukin (IL)-6 were increased by OSM. OSM affected mesenchymal cell differentiation and promoted the hematopoietic supportive activity of marrow stromal cell lines. As OSM production is induced by cytokines from hematopoietic cells, OSM may be a key factor in mutual regulation between hematopoietic cells and stromal cells in the bone marrow. OSM may play a role as a regulator in maintaining the hematopoietic microenvironment in marrow by coordinating mesenchymal differentiation.

Adipocytes↗