Analysis of function of NMDA receptor using genetically engineered mice by conditional mutagenesis
| Project/Area Number |
15500277
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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 |
Neurochemistry/Neuropharmacology
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| Research Institution | NATIONAL INSTITUTE FOR BASIC BIOLOGY (2004)
Okazaki National Research Institutes (2003) |
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Principal Investigator |
SASAOKA Toshikuni National Institute for Basic Biology, Center for Transgenic Animals and Plants, Associate Professor, 形質転換生物研究施設, 助教授 (50222005)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Torahiko National Institute of Neuroscience, NCNP, Section Chief, 神経研究所・疾病研究第七部, 室長 (90171785)
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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: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | genetically engineered mice / conditional mutagenesis / NMDA receptor / recombinant antibodies / phage display / amino acid substitution / 神経症状 |
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| Research Abstract |
To understand molecular mechanism causing the neurological phenotype through an activation of the N-methyl-D-aspartate receptor (NMDAR), we generated and analyzed mutant mice harboring activated NMDAR. We developed a conditional mutagenesis in mice to enable substitution of a critical sequence in NMDAR activation in a neuron-specific manner for purposes of functional analysis. We created mice carrying a tandem array of a normal exon and a mutated exon, separated by an artificial intron, in which the normal exon could be exclusively expressed. The normal exon was deleted by Cre-loxP recombination, allowing the alternative expression of the mutant exon. We successfully generated the mutant mice harboring an aberrant NMDAR activation by a substitution of a critical amino acid in the second trans-membrane domain of the NMDAR, and the mutant mice exhibited a clasping of the limbs.
To block the clasping phenotype of the mutant mice we screened NMDAR agonists, NMDAR antagonists and several dru
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gs for Parkinson's disease and found a non-competitive NMDAR antagonist completely blocked the clasping phenotype.
We tried to develop a specific antibody recognizing the substituted amino acid of the NMDAR and obtained a rabbit polyclonal antibody recognizing the second trans-membrane domain of the NMDAR. The specificity of the polyclonal antibody recognizing the substituted amino acid is still being examined.
We applied a recombinant antibody technology to develop a specific antibody against the substituted amino acid of the NMDAR. We established a simple, rapid and highly efficient ligation-transformation method for unidirectional subcloning to generate far larger numbers of transformants than previous procedures, and constructed single chain Fv phage-display libraries with a large-scale and a high-complexity consisting of approximately 1 x 109 independent clones. So far a recombinant antibody with a high affinity was obtained from the library. An antibody against the substituted amino acid of the NMDAR is being screened. Less
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Report
(3 results)
Research Products
(12 results)
