| Project/Area Number |
11670053
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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 physiology
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| Research Institution | Kansai Medical University |
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Principal Investigator |
OMORI Koichiro Kansai Med. Univ.,Dept.of Physiol.,Assistant Prof., 医学部, 助教授 (80094465)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Akitsugu Kansai Med. Univ., Dept.of Physiol., Lecturer, 医学部, 講師 (30174775)
MATSUDA Hiroko Kansai Med. Univ., Dept.of Physiol., Professor, 医学部, 教授 (10181736)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | inwardly rectifying K+channel / PC12 cell / cultured hippocampal neuron / adenovirus vector / 海馬神経細胞 |
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| Research Abstract |
The inwardly rectifying K+ channels have been found in a variety of cell types including cardiac myocytes and neuronal cells, and play diverse cellular functions such as maintenance of the resting conductance, pacemaker activity, synaptic inhibition, and neuronal firing rates. IRK1, one of the classical inwardly retifying K+ channel, has long C terminal domain comprised of 〜250 amino acidsand the function of the domain has not been elucidated.
To analyze the function, we transfected PC12 cells with IRK1 and GFP genes and treated with NGF to differentiate to neuronal cells. Cultured hippocampal neurons from rat fetus were also transfected with the same genes. The gene products could be detected by Western blotting in the PC12 cells but not in the clutured hippocampal neurons. We examined the localization and distribution of the IRK1 gene products by immunohistochemistry in the transfected PC12 cells. The immunoreactivity appeared to be localized mainly in plasma membrane and to a lower extent, in other organelles, which could not been confirmed clearly because of the low expression levels. Then, we used adenovirus vector to introduce IRK1 gene to the cells. IRK1 gene was inserted into the adenovirus vector and the created recombinant adenovirus DNA was used to transfect HEK293 cells to make the recombinant virus particles. The titer of the recombinant virus particles were 〜5x105 pfu/mL.. We used the virus particles to infect PC12 cells and cultured hippocampal neurons at various MOI, but could not get high expression levels owing to low virus titer and high toxicity of the virus solution. We tried to purify the virus particles to remove the toxicity and to get virus with high titer. However, the virus titer was decreased quickly and inactivated during the purification. We suppose the inserted IRK1 gene itself may inhibit the recombinant virus activity.
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