| Project/Area Number |
08044238
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Akita university School of Medicine |
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Principal Investigator |
MIURA Naoyuki Akita University, School of Medicine, Associate Professor, 医学部, 助教授 (40165965)
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| Co-Investigator(Kenkyū-buntansha) |
RAUSCHER FRA ウィスター研究所, 分子腫瘍学, 助教授
YOSHIDA Nobuaki Research Institute, Osaka Medical Center of Maternal and Child Health, Director, 部長 (10250341)
RAUSCHQR Frank Jr.III The Wistar Institute, Associate Professor
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| Project Period (FY) |
1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1996: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | MFH-1 gene / Knockout mice / Kidney / development / monoclonal antibody / MFH-1 protein |
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| Research Abstract |
We isolated the MFH-1 gene as one of the forkhead gene family in 1993. The family are growing and more than 20 genes are known in mice. The MFH-1 gene is expressed at the somitic mesoderm and later expressed strongly in the sclerotome. It is also expressed in the developing metanephros and later on in the glomerulus after birth.
We made knockout mice of the MFH-1 gene by homologous recombination in the ES cells. First, we cloned the mouse genomic MFH-1 gene and determined its genomic structure. We found that it has no introns. We made a targeting vector by replacing the exon with neo-resistance cassette and introduced it into ES cells by electroporation. Out of 480 neomycin- and gancyclovir-resistant clones, 6 were identified to be homologous recombinants. We made chimera mice by injecting them into a blastocyst and obtained heterozygotic mice by mating chimera mice and wild B6 mice. The heterozygotes were asymptomatic. However, homozygotes were dead within ten minutes after birth.
Homozygotes had abnormalities in several tissues. Here we focused the phenotype of kidneys. The size of kidneys in homozygotes was half that of the wild-type. Histologically the glomerulus, renal tubular cells and blood vesssels were present normally. But dilated renal calces were seen in homozygotes.
Next, we cloned the human genomic MFH-1 gene and determined its nucleotide sequence. We found that human MFH-1 protein is 501 amino acids long and the amino acid sequence of the forkheah domain is identical in human and mouse. Identity of the mouse and human MFH-1 protein is 94%.
We also made monoclonal antibodies against the human MFH-1 protein. Using thses antibodies, we found that the MFH-1 protein is expressed in chondrosarcoma, osteosarcoma and Wilms tumor cell lines.
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