PubMed HealthSearch

Biomedical subjects

T Sasado

Publications and source records attributed to T Sasado.

4 recordsLinked to original sources

Expression of murine early embryonic antigens, SSEA-1 and antigenic determinant of EMA-1, in embryos and ovarian follicles of a teleost medaka (Oryzias latipes).

Stage-specific embryonic antigen-1 (SSEA-1) and the antigenic determinant of monoclonal antibody EMA-1 are expressed in a stage-specific manner in mouse early embryos. To study whether these antigens generally exist in fish, expression of the antigens was examined in embryos, ovarian follicles, and adult tissues of a teleost medaka (Oryzias latipes), using immunohistochemical techniques. In 1-cell-stage embryos, these carbohydrate antigens were found in numerous cytoplasmic granules in the blastodisc and the cortical cytoplasm. These granules gradually decreased in number as the embryos developed. In 4-cell-stage embryos, the antigens appeared on the cleavage planes and were located on the cleavage planes within the blastoderm in the following cleavage stages. In blastula-stage embryos, the expression was ubiquitously found on the cell surface of blastomeres. At the mid-gastrula stage, the antigens were restricted to the enveloping layer, yolk syncytial layer, and cortical cytoplasm, but were rarely found in deep cells that contribute to formation of the embryonic body. In later-stage embryos and adult fish, the antigens were located in various tissues. In ovarian follicles, the antigens were found in granules of oocytes and granulosa cells. These observations were basically consistent with those in mice; however, expression in 1-cell-stage embryos and ovarian follicles has not been observed in mice. This unexpected finding suggests that the antigens are produced in granulosa cells and transferred to 1-cell-stage embryos via oocytes, and that the antigens involved in the early developmental process are maternally prepared in teleosts.

Animals

Usefulness of the medaka beta-actin promoter investigated using a mutant GFP reporter gene in transgenic medaka (Oryzias latipes).

The activity of the medaka beta-actin promoter as a ubiquitous expression vector in transgenic medaka was examined using complementary DNA of the green fluorescent protein (GFP). Plasmid pOBA-GFP contained both the medaka beta-actin promoter and cDNA of the wild-type GFP, while pOBA-hGFP contained the medaka beta-actin promoter and cDNA of the mutant GFP in which serine was substituted for threonine at position 65 and codon usage was humanized to promote translation in vertebrate cells. The ApaI-SmaI fragment of both plasmids was microinjected into the nuclei of oocytes or the cytoplasm of embryos at the one-cell stage. The gene expression was detected, using a fluorescent stereomicroscope, from early stages of development to 1 week after hatching. The expression of the wild-type GFP was detected in early embryos, in the yolk sac and in small portions of the muscle and epidermis. This expression pattern was similar to that of the Escherichia coli beta-galactosidase reporter gene (lacZ), driven by the medaka beta-actin promoter, which was examined in our previous studies. The mutant GFP was expressed in early embryos and in many tissues such as the epidermis, blood vessels, muscle, notochord, fin ray, gut, eyes, and yolk sac, and the fluorescence was much stronger than that of the wild-type GFP. Thus, the usefulness of the medaka beta-actin promoter as a ubiquitous expression vector was confirmed using the mutant GFP as a reporter gene.

Actins

Establishment of a pluripotent cell line derived from a medaka (Oryzias latipes) blastula embryo.

A pluripotent cell line, OLES1, was established from a blastula embryo of a small freshwater fish, medaka (Oryzias latipes). Cells of this cell line were small and round, and they grew actively and stably in culture as dense clusters. They exhibited a positive alkaline phosphatase activity upon histochemical staining. When the cells were treated with retinoic acid, differentiation into various types of cells, including melanocytes, dopa-positive precursors of melanocytes, and cells with a molecular marker of skeletal muscles, troponin T, was induced in vitro. The present study opens a way to establishing embryonic stem cell lines in fish.

Alkaline Phosphatase

An efficient expression vector for transgenic medaka construction.

The transparency and external fertilization of the eggs of medaka (Oryzias latipes) make them ideally suitable for investigating molecular interactions that occur during vertebrate development. Genetically engineered medaka is a potential tool for such studies. It requires several types of suitable expression vectors. To obtain abundant and ubiquitous expression of foreign genes in medaka embryos, we have designed an expression vector that contains the proximal promoter and enhancer elements and polyadenylation signal of the medaka beta-actin gene. The utility of this "all-medaka" expression vector was examined using the Escherichia coli lacZ gene as a reporter gene. Most of the injected embryo showed high gene expression, and several embryos showed ubiquitous expression even at six days after injection. Of nine individuals derived from the injected embryos and grown until adult stage, one produced expression-positive F1 fish. The transgene was identified in these F1 using polymerase chain reaction (PCR). These data revealed that the expression vector based on the expression cassette from the medaka beta-actin gene should be useful for making transgenic medaka. The cloned gene in this cassette vector is stably transmittable and efficiently expressible.

Actins