| Project/Area Number |
10670008
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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 |
General anatomy (including Histology/Embryology)
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SAGARA Hiroshi Inst. Medical Sci., The University of Tokyo, Research Associate, 医科学研究所, 助手 (50145041)
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| Co-Investigator(Kenkyū-buntansha) |
KATSUKI Motoya Inst. Medical Sci., The University of Tokyo, Professor, 医科学研究所, 教授 (20051732)
HIROSAWA Kazushige Inst. Medical Sci., The University of Tokyo, Professor, 医科学研究所, 教授 (30009980)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | RPE65 / Vitamin A / Retinal pigment epithelium / gene targeting / Zebrafish / Drosophila melanogaster / ノックアウトマウス / ジーンターゲッティング / 網膜色素上皮 / 単クローン抗体 / isomerohydrolase |
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| Research Abstract |
RPE65 is a molecule specifically expressed in the retinal pigment epithelial cells. To clarify the function(s) served by the RPE65, following studies directing toward the gene targeting were performed.
1. Trial to analyze knock out mice.
PCR fragments were amplified using PCR primers designed from the nucleotide sequence of rat RPE65 and the genomic DNA from mouse ES cells as a template. Nucleotide sequences of the fragments had high homology with those of known animals. During this study, a report concerning the RPE65 knock out mice to prove that the mouse models were insufficient for the aim of our study. So we decided to concentrate on the following studies.
2. cDNA cloning and analysis of developmental expression of the zebrafish RPE65
The cDNA clones of the zebrafish RPE65 were isolated using degenerate PCR and cDNA library screening methods. Expression patterns of the RPE65 transcripts during embryonic development were analyzed by in situ hybridization methods. The RPE65 transcripts were first detected in the epiphysis at 12-13 hours post fertilization (hpf). Expression in the eye was observed only after 42-45 hpf, indicating the early requirement of the RPE65 in the epiphysis.
3. Identification of the RPE65 ortholog of the fruit fly, Drosophila melanogaster
Fragments of the nucleotide sequences having high homology with those of chick RPE65 were obtained by screening the Drosophila genomic DNA database. The cDNA clones were isolated and the expression patterns were analyzed with Northern blot analysis. The transcripts were detected only in the head and not in the other parts of the body. In situ hybridization analysis is now on the way to identify the expressing cells.
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