| Project/Area Number |
02454091
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
畜産学(含草地学)
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| Research Institution | The Institute of Medical Science, The University of Tokyo |
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Principal Investigator |
TOYODA Yutaka Inst. Med. Sci., Univ. Tokyo, Professor, 医科学研究所, 教授 (90050418)
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| Co-Investigator(Kenkyū-buntansha) |
KATAGIRI Takuya Inst. Med. Sci. Univ. Tokyo, Assis. Prof., 医科学研究所, 助手 (70126100)
NAITO Kunihiko Inst. Med. SCi. Univ. Tokyo, Assis. Prof., 医科学研究所, 助手 (20188858)
KYUWA Shigeru Inst. Med. Sci. Univ. Tokyo, Assis. Prof., 医科学研究所, 助手 (30177943)
甲斐 知恵子 東京大学, 農学部, 助教授 (10167330)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1991: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1990: ¥5,400,000 (Direct Cost: ¥5,400,000)
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| Keywords | gamete / embryonic stem cell / transgenic animal / in vitro fertilization / chimeric embryo / germ line chimera / gene expression / embryo transfer / 透明帯 / カルシウム / キメラ / 卵子 / 配偶子 / 胚盤胞 |
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| Research Abstract |
1. Oocyte maturation and fertilization in vitro.
In order to establish the optimum condition for manipulating gametes and embryos, various factors affecting oocyte maturation and fertilization were investigated in vitro. We have found that porcine oocytes matured in porcine follicular fluid have normal developmental ability and they showed essentially the same fluctuation pattern of histone Hl kinase activity during the course of meiotic maturation. It was also found that the in vitro fertilization of cumulus-free mouse oocytes was improved significantly by increasing calcium concentration in the fertilization medium.
2. Expression of exogenous genes in preimplantation embryos.
DNA carrying the SV40 early promoter fused with the Escherichia coli beta-galactosidase gene, (lacZ) was microinjected into the pronucleus of in vitro fertilized mouse eggs and the expression was detected by staining embryos with X-gal. It was found that the exogenous gene was expressed from the 4-cell stage and reached the maximum at the morula stage. All of the embryos have shown a mosaic pattern of staining, suggesting unequal distribution on injected DNA and/or early differentiation of blastomeres.
3. Establishment of new ES cell lines.
We have established two ES cell lines from the blastocysts of 129/SvJ mice. The cells from one cell line (A3-1) were demonstrated to have pluripotency, including the ability to develop to germ line chimeras following injection into the host blastocysts. The cells have XY sex chromosomes and are homozygous for the albino locus. We further found that the efficiency of isolation of ES cells was markedly improved when the blastocysts were cultured overnight for hatching and then transferred on the feeder cells.
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