2004 Fiscal Year Final Research Report Summary
Generation of transgenic mice expressing alphalA-calcium channel protein with highly expanded polyglutamine tract.
| Project/Area Number |
15590881
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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 |
Neurology
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
ISHIKAWA Kinya Tokyo Medical and Dental University, Department of Neurology, Assistant Professor, 医学部附属病院, 講師 (30313240)
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| Project Period (FY) |
2003 – 2004
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| Keywords | cerebellum / genetics / neuron / ataxia / degeneration |
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| Research Abstract |
The most important aim of this study is to generate transgenic mice that express human alpha1A-calcium channel protein with highly expanded polyglutamine tract to gain model mice that could be a n indispensable material for exploring pathogenesis of SCA6, an autosomal dominant cerebellar ataxia caused by a mild expansion of polyglutamine in alpha1A-calcium channel. To accomplish this, we cloned human alpha1A-calcium channel cDNA which contains a CAG repeat tract encoding 165 polyglutamines. This clone was inserted downstream of the Purkinje cell specific promoter. Then the construct was microinjected into C57B16/J mouse strain. We obtained 200 mice by two microinjections, and screened by PCR amplification of genomic DNA, whether any of these mice harbor human alpha1A-calcium channel construct. However, we did not observe such mice. Knowing that similar difficulties could be overcome by changing mice strain to Fbv, we next underwent microinjection to the Fbv mice strain. These experimen
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ts were done on September 2004. After screening mice DNA for positive injection by aforementioned procedure, we found on December 2004 that two FO mice properly harbor human alpha1A-calicum channel cDNA construct. These mice were backcrossed with wild type Fbv mice. By the end of March 2005, we have screened several mice of F1 generation. However, we did not observe evidence for germline transmission. We will continue backcrossing FO mice with wild -type mice.
We also planned to analyze SCA6 patients' brains whether the expanded polyglutamine induces proteolytic cleavage of alpha1A calcium channel as suggested by our preliminary in vitro study. For this aim, we generated rabbit polyclonal antibody against the polypeptide near the polyglutamine tract. The specificity of this antibody was assessed by SDS-PAGE analysis on recombinant channel protein. By using this antibody, we found that the polyglutamine aggregates seen in SCA6 Purkinje cells indeed contain alpha1A-calcium channel. We also observed that large aggregates identified by the antibody against the Carboxyl-terminal peptide are formed by cluster of polyglutamine aggregates. These findings suggested that the human alpha1A-calcium channel protein is indeed processed in Purkinje cells. Processed fragments are likely to be prone to aggregatie formation Less
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