| Project/Area Number |
05454655
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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 |
Developmental biology
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| Research Institution | National Institute of Genetics |
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Principal Investigator |
NAKATSUJI Norio National Institute of Genetics, Mammalian Development Laboratory, Professor, 遺伝実験生物保存研究センター, 教授 (80237312)
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| Co-Investigator(Kenkyū-buntansha) |
SHIRAYOSHI Yasuaki National Institute of Genetics, Mammalian Development Laboratory, Assistant Prof, 遺伝実験生物保存研究センター, 助手 (90249946)
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1995: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1994: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1993: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Keywords | Primordial germ cells / Fatal germ cells / gp130 / EG cell lines / Gene transfection / Apoptosis / Tunor necrosis factor / Retinoic acid / TNF-α / 生殖細胞 / マウス / 細胞増殖 / 増殖因子 / 発生工学 / TNF-alpha |
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| Research Abstract |
1. Primordial germ cells (PGCs) show proliferation and growth arrest in culture. Such growth pattern could be regulated autonomously or by somatic cells. The mixed culture experiments using PGCs from 8.5 and 11.5 dpc embryos indicated no detectable interaction between the PGCs and somatic cells at the two stages. Clonal culture of PGCs did not reveal any subpopulation of PGCs in terms of growth rate and morphological changes. We conclude that there is an autonomous regulation of growth, which is not strictly timed by the number of cell divisions.
2. Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse PGCs in culture. We have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. A combination of IL-6 and soluble IL-6 binding subunit (sIL-6R) fully reproduced the LIF action on PGCs. Addition of neutralizing antibody against gp130 in culture remarkably blocked PGC survival. These results suggest the pivotal role of gp130 in PGC development. We have further demonstrated that a combination of LIF with forskolin or retinoic acid caused continuation of the proliferation of PGCs.
3. We tested transfection methods of electroporation, liposome-mediated transfection and calcium phosphate (CaPO4) co-precipitation method. When PGCs were transfected with plasmid pSV-LT by CaPO4 co-precipitation method, transient expression of simian virus 40 large-T antigen was detected in an average of 18% of PGCs transfected. Transient expression of adenovirus 2 E1B 19kDa protein in about 15% of PGCs significantly retarded disappearance of PGCs from the culture system and increased the number of rounded PGCs.
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