| Project/Area Number |
09558107
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Laboratory animal science
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| Research Institution | Kumamoto University |
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Principal Investigator |
ARAKI Kimi Kumamoto University School of Medicine, Assistant Professor, 医学部, 助手 (90211705)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Misao Kumamoto University, Center for Animal Resources & Development, Associate Professor, 動物資源開発研究センター, 助教授 (60253720)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥10,800,000 (Direct Cost: ¥10,800,000)
Fiscal Year 1999: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1998: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1997: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | Cre / lox system / microinjection / transgenic mice / targeted integration / Ioxシステム / lox システム / 部位特異的遺伝子導入 / Cre-loxシステム |
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| Research Abstract |
A transgenic mice line carrying a single copy of the transgene that is CAG promoter + lox71+bsr gene was established, and the homozygous male mice were produced through intercrossing. Fertilized eggs were collected from superovalated females crossed with the homozygous male mice, and injected with the Cre expression vector, pCAGGS-Cre, and the targeting vector that is lox66+NLSlacZ+MC1neo+pBluescript. When the targeting vector was inserted into the lox71 site in the mouse genome, the embryos were stained into blue with X-gal because the NLSLacZ gene was placed under the CAG promoter. The injected eggs were transferred into the oviducts of foster mothers, and the embryos were collected at 12.5 dpc, stained with X-gal. At the same time, the genomic DNAs were extracted from the placentas, and the integration pattern of the targeting vector was examined with PCR and Southern blotting. About 3500 eggs were microinjected under various conditions. Out of 486 embryos carrying the transgene, 5 embryos showed positive X-gal staining, demonstrating 1% of targeting efficiency. The DNA analyses of these X-gal positive embryos confirmed the targeted integration. However, the efficiency of 1% is not enough for effective production of transgenic animal. We are going to improve the system and raise the efficiency of targeting integration.
We have also produced transgenic mice lines carrying a lox71 site as promoter resources by gene trapping method. Until now, 17 mice lines have been established and analyzed the expression pattern of transgene.
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