| Project/Area Number |
14370052
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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 | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY |
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Principal Investigator |
KATO Ichiro Toyama Medical and Pharmaceutical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (50250741)
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| Co-Investigator(Kenkyū-buntansha) |
HIRAGA Koichi Toyama Medical and Pharmaceutical University, Faculty of Medicine, Professor, 医学部, 教授 (40004733)
NISHIJO Hisao Toyama Medical and Pharmaceutical University, Faculty of Medicine, Professor, 医学部, 教授 (00189284)
KONDO Takeo Tohoku University Hospital, Assistant Professor, 助手 (30282130)
武田 正利 富山医科薬科大学, 医学部, 助手 (70345578)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥13,700,000 (Direct Cost: ¥13,700,000)
Fiscal Year 2004: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2003: ¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 2002: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Keywords | glycine / H protein / ES cells / Cre recombinase / knockout / mouse / prenatal death / encephalopathy / Cre レコンビナーゼ |
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| Research Abstract |
1.We made the knock-out vector (H protein gene-KO) that carry mouse H protein 5'-genomic DNA, loxP, exon1, loxP, pGK-neo, loxP, mouse H protein 3'-genomic DNA in this sequence.
2.We introduced the linearized knock-out vector into mouse ES cells and got 11 cell lines of ES cells that have undergone homologous recombination between the ES genome and knock-out vector.
3.Next, we microinjected recombinated ES cells to C57 Black mouse blastocysts and obtained 7 male chimeras, 3 of them have undergone germ-line transmission of the mutated allele.
4.We then mated F1 mice with Cre recombinase-expressing transgenic mice. In the next generation, excision of exon1 DNA sequence was confirmed by genome PCR and Southern blot analyses.
5.To determine whether the H protein expression is downregulated in the tissues of H protein +/-mice, we performed Western blot analysis using anti-H protein rabbit polyclonal antibody. Western blot analysis indicated that the H protein is decreased to 50% in the brains of +/-mice as compared to +/+ mice.
6.We mated males and females of H protein +/-mice and analyzed the genotypes of newborn pups and embryos. PCR analyses have clearly shown that homozygous H protein knockout mice can not be born alive or even develop to E14.
7.In the present study, we obtained the H protein +/-mice that have reduced (50%) levels of H protein and showed that H protein is essential for normal development and survival of mouse embryos. By using Cre/lox P system, we will be able to know the unknown functions of H protein in vivo.
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