| Co-Investigator(Kenkyū-buntansha) |
ADACHI Shinji Hokkaido Univ., Faculty of Fisheries Science, Professor, 大学院水産科学研究院, 教授 (40231930)
YOSHIZAKI Goro Tokyo Univ.of Marine Science and Technology, Associate Professor, 海洋科学部, 助教授 (70281003)
HIRAI Toshiro Teikyo Univ.of Sci. and Tech., Div.of Advance Science and Technology., Lecturer, 理工学部, 講師 (30238331)
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| Research Abstract |
This project was planned for development of foundational technology of surrogate production in teleost fish. The surrogate production in fish is a technique to get the gametes of some genotype through the gonad of other genotype. We got the following results in the past three years.
1)When artificial mRNA, combined with green fluorescence protein (GFP) and zebrafish nos1 3'UTR, was injected into fertilized eggs in herring, goldfish, loach, pearl danio, ice goby, ukigori goby, medaka, verfin flounder and eel, primordial germ cells (PGCs) were visualized during embryonic development.
2)Primordial germ cells (PGCs) were visualized during embryonic development in herring, goldfish, loach, pearl danio, ice goby, ukigori goby, medaka, verfin flounder and eel by injecting artificial chimeric mRNA combined with green fluorescence protein (GFP) and zebrafish nos1 3'UTR into fertilized eggs.
3)A method for single PGC transplantation was developed and named SPT method. In this method, visualized PGC
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s with GFP were isolated from early somitogenesis stage and transplanted into the blastulae in which differentiation of endogenous PGC was suppressed by injecting morpholino oligo (MO) nucleotide designed to maternally-supplied dead end (dnd) mRNA.
4)Fertilized eggs and viable offspring of pearl danio, inter-specifically different from zebrafish, were obtained from natural spawning of zebrafish parents developed from blastula to which PGC from pearl danio embryo were transplanted. Functional sperm of goldfish or loach belonging to a different genus or family from that of zebrafish, respectively, were also differentiated in the zebrafish host developed from blastulae to which PGC from each species were transplanted.
5)Androgenetic embryo between loach egg and goldfish sperm was lethal before epiboly. The blastomeres of this cybrid, however, were survived in the host embryos, when they were transplanted into the loach or goldfish blastulae. PGCs from this lethal cybrid were also detected in the host embryos of goldfish or loach.
6)Functional gametes were differentiated from PGCs isolated from cryopreserved genital anlage taken from hatched fry and transplanted peritoneal cavity of host fry. All rainbow trout offspring were also obtained from the triploid masu salmon of which PGC from rainbow trout were transplanted into peritoneal cavity of hatched fry.
7)In rainbow trout, functional gametes were differentiated from gonium, when they were isolated from developed gonads and transplanted into peritoneal cavity of hatched fry. Spermatogonia and oogonia were differentiated to egg and sperm, respectively, in the female and male host. Less
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