|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1994 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Medaka (Oryzias latipes), a small freshwater fish, is known as a good experimental model for study of genetic processes involved in vertebrate development. Gene targeting has been developed in mice and has achieved grate success for disrupting the function of a desired gene. This technique, however, has not yet been developed in any fish species. In this project, establishment of ES cells and chimera formation using the ES cells were tried in order to develop gene targeting system in medaka.
A pluripotent cell line, OLES1, was first established by culturing blastodermal cells from a blastula embryo of an inbred wild type strain, HNI-I.The cells were small and round in shape, and grew actively and stably in culture as dense clusters. They exhibited a positive alkaline phosphatase (AP) 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 w
ith a molecular marker of skeletal muscles, troponin T,was induced in vitro. The second cell line, OLES2, with similar morphology to that of OLES1 and an positive AP activity was also established. 85% of the cells of this cell line showed a normal diploid chromosome unmber, 48. LIF has any apparant effect on the growth of this cell line in a range of concentrations from 0 to 10^4/ml. Growth of the cells were, however, apparently promoted by bFGF depending on the concentrations up to 20ng/ml. The population doubling time of the cells at this concentration of bFGF was 24 hous.
Chimera formation have been tried by injecting OLES1 cells into blastula embryos of an albino strain. However, any apparent chimeras with melanocytes in the body or germ-line chimeras have not been produced yet. OLES1 cells with a fluorecent dye were injected into the embryos to trace their behavior in one day-old embryos. An wide distribution of the cells was observed in all kinds of embryonic tissues by a histological examination. Less