Embryonic stem cell differentiation system for evaluating gene functions involved in physiological megakaryocytic differentiation.

Megakaryocytic differentiation is accompanied by marked morphological changes induced by endomitosis and proplatelet formation. Molecular mechanisms underlying this unique cell differentiation process have been investigated by gain/loss-of-function studies using leukemic cell lines. However, these cell lines cannot completely mimic physiological megakaryocytic differentiation, including the morphological changes, and sometimes lead to contradictory results between cell lines. The goal of this study was to establish a novel cell differentiation system that completely mimics physiological megakaryocytic differentiation for analyzing gene function. To that end, we used homologous recombination to prepare an embryonic stem (ES) cell line containing a GFP-transgene driven by the PF4 promoter at the Hprt locus. Differentiation of these cells resulted in megakaryocytes and proplatelets, suggesting physiological megakaryocytic differentiation. However, the number of GFP-expressing cells was low (1.7% GFP(+) cells among CD41(+) cells). Insertion of full-length or small core ß-globin insulators on either side of the transgene significantly increased the number of GFP-expressing cells (∼60% GFP(+) cells among CD41(+) cells), and GFP-expression was specifically observed in megakaryocytic cells. Similar results were obtained with other ES cells containing a GPIIb-GFP transgene. Altogether, we have succeeded in efficiently expressing exogenous genes specifically in differentiating megakaryocytes and in establishing a novel ES cell differentiation system for analyzing gene function involved in physiological megakaryocytic differentiation.

Annealing effects on electrical properties of pure and tin-doped indium oxide thin films.

The annealing effects on the properties of ITO and pure In2O3 thin films have been investigated. The thin films were deposited with various O2 flow ratios to total gas flow by pulsed dc magnetron sputtering. The post-deposition annealing of the thin films was carried out for 30 minutes at various temperatures ranging up to 500 degrees C in air. It was found through the comparison of the carrier density of ITO and In2O3 thin films that the carrier electrons of the ITO thin films came from both of the dopant Sn and oxygen vacancies under the annealing less than 400 degrees C. Therefore, the ITO thin films deposited with lower O2 flow ratio exhibited higher carrier density due to many oxygen vacancies; in consequence, they exhibited lower resistivity at the annealing up to 400 degrees C. On the other hand, the carrier density of ITO thin films was almost identical regardless of O2 flow ratio when they were annealed at 500 degrees C. This fact indicates that most carrier electrons of the ITO thin films were brought by the dopant Sn at the annealing temperature of 500 degrees C. However, the ITO thin films deposited with lower O2 flow ratio exhibited higher Hall mobility; as a result, they showed lower resistivity at the annealing of 500 degrees C. Atomic force microscope, X-ray diffraction and X-ray reflectivity measurements revealed that the ITO thin films deposited with lowe O2 flow ratio exhibited dense structure even after they were annealed at 500 degrees C. Hence, the carrier electrons of the dense ITO thin films deposited with low O2 flow ratio can conduct better, as a result, the ITO thin films exhibited high Hall mobility and low resistivity.

Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

New protein purification system using gold-magnetic beads and a novel peptide tag, "the methionine tag".

Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.