| Project/Area Number |
06670132
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General pharmacology
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| Research Institution | National Children's Medical Research Center |
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Principal Investigator |
NISHIMURA Chihiro National Children's Medical Research Center,Department of Pediatric Pharmacology, laborrtory Chief, 小児医療研究センター・小児薬理研究部, 室長 (70150571)
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| Co-Investigator(Kenkyū-buntansha) |
KOKAI Yasuo National Children's Medical Research Center, Department of Pathology, Laboratory Chief, 病理学教室, 助手 (20178239)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | aldose reductase / gene targeting / diabetic complications / 酵素欠失マウス |
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| Research Abstract |
Aldose reductase (alditol : NAD(P)+ 1-oxidoreductase) is an enzyme implicated in the pathogenesis of various diabetic complications. To elucidate the physiological function as well as the molecular, mechanisms underlying the involvement of this enzyme in the development of diabetic complications, we planned to 1) generate the mice lacking the enzyme by gene targeting and to 2) express human enzyme in these mice.
To construct targeting vectors to knockout aldose reductase (AR) gene, genomic fragments of mouse AR gene was required. Among the initial 25 clones obtained from mouse genomic library, none of the clones was identical to the mouse vas deferens protein, previously reported as the mouse counterpart of AR. We therefore newly screened the mouse kidney cDNA Iibrary using rat cDNA probe. The isolated clone encoded a 316-amino acid protein with 97 % identity to rat lens AR and 69 % identity to the mouse vas deferens protein. RNA blot analysis demonstrated abundant expression of the enz
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yme transcript in the testis, skeletal muscle and kidney. The isolated cDNA was expressed in Escherichia coli and the recombinant protein was purified to homogeneity by affinity chromatography and chromatofocusing. The expressed enzyme demonstrated the typical characteristics of AR previously reported for the enzyme protein isolated from other animal species. These results indicated the presence of a closely related subgroup within the aldo-keto reductase superfamily in mouse tissues. Based on the sequence data obtained from the cDNA clone, PCR primers were synthesized to identify the genomic clone of mouse enzyme among the former 25 isolated clones. Two of the clones were demonstrated to contain intron structure. Analysis of the restriction enzyme map and the sequences of these genomic clones indicated that mouse AR gene spans approximately 14 kb and encoded by 10 exons. The boundaries between exons and introns are determined by comparing the genomic sequence to cDNA sequence. All of the splice junctions of the gene conformed with the GT splice donor and AG splice acceptor rule. Presently we are making targeting vectors to generate knockout mice by use of the genomic fragments isolated from this gene. Less
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