| Project/Area Number |
15570145
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Kumamoto University |
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Principal Investigator |
ARAKI Masatake Kumamoto University, IRDA, Division of Bioinformatics, Associate Professor, 生命資源研究・支援センター, 助教授 (80271609)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHINOBU Kumiko Kumamoto University, IRDA, Division of Bioinformatics, Assistant Professor, 生命資源研究・支援センター, 助手 (20274730)
ARAKI Kimi Kumamoto University, IMEG, Division of Developmental Genetics, Associate Professor, 発生医学研究センター, 助教授 (90211705)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Rhoa / gene trap / small GTPase / geo / Knock-in mouse / ジーントラップ / ノックアウトマウス |
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| Research Abstract |
We have developed the exchangeable gene trap system. The reporter gene, β-geo, can be replaced into any other DNA of interest through Cre-mutant lox site-specific recombination.
The Ayu17-52 mouse line is the Rhoa gene knockout mouse line produced by the exchangeable gene trap method. Rho family proteins are Ras-related small GTPases that regulate a variety of cellular processes. Its function in vivo is still unclear.
The purpose of this research project is to clear up the functional compatibility of Rho family genes in vivo.
Heterozygous β-geo(Rhoa^<+/geo>) mice showed severe growth retardation, and most of them died in a few weeks after birth.
At the first step, Rho1 gene of yeast, one of homologues of mouse Rhoa, was knocked in the trap allele instead of the β-geo gene. Heterozygous Rho1 knock in mice showed no growth retardation. At the second step, human skeletal muscle α actin(HSA) gene promoter was knocked in the trap allele instead of the β-geo gene. At the third step, mouse Rhoc cDNA was knocked in the trap allele instead of the β-geo gene. These replacements could rescue the phenotype of heterozygote. Finaly, we have tested a replacement of reporter gene from β-geo to EGFP.
After replacement, the change of the phenotype was observed, too. The phenotype was disappeared completely in heterozygous EGFP(Rhoa^<+/BGFP>) mice. Since the both of β-geo and EGFP gene are expressed quite strongly and ubiquitously, the strong expression of β-geo protein might affect the phenotype severely.
Homozygous EGFP(Rhoa^<EGFP/EGFP>) mice show embryonic lethality like as homozygous Rho1 knock in (Rhoa^<Rho1/Rho1>) mice. It means that this is the phenotype of Rhoa protein deficiency. Now, we are investigating if Rhoc replacement might rescue the phenotype of homozygote.
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