1994 Fiscal Year Final Research Report Summary
Developmental Study on a Real-time Measurement of Gene Transcriptional Activity by Means of lon-channel Reporter Genes.
| Project/Area Number |
05557003
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General physiology
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| Research Institution | FACULTY OF MEDICINE,UNIVERSITY OF TOKYO |
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Principal Investigator |
TAKAHASHI Kunitaro Faculty of Medicine, University of Tokyo, 医学部(医), 教授 (10010034)
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| Co-Investigator(Kenkyū-buntansha) |
OKAMURA Yosushi Faculty of Medicine, University of Tokyo, 医学部(医), 助手 (80201987)
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| Project Period (FY) |
1993 – 1994
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| Keywords | Transcriptional Activity / Reporter Gene / Ion Channels / Non Invasive / Ascidian Embryos / Isolated Blastomere / Ascidian Na Channels / RNA Injection |
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| Research Abstract |
Regulation of transcriptional activities of a gene has been analyzed by means of various reporter genes linked to 5' upper stream of the coding region of the gene. The reporter genes generally used are beta-Gal, CAT,Luciferase genes, which are detected by the enzymatic activities in homogenates or by histochemical methods after fixation. In order to analyze transcriptional activities in living cells ion channel genes are extremely suitable because activities of ion channels can be measured electrophysiologically in living state and sometimes may be detected optically through changes in intracellular ion concentrations by appropriate fluorescent indicators. Aim in the present project is to test the possibility of using ion channel genes as reporter genes in the model neural induction system of the ascidian two-cell pair. Obtained results are : (1) In order to use an ascidian Na channel gene as a reporter we cloned TuNa I from larval cDNA library and whole nucleotide sequence was determined. The insensitivity to TTX was suggested to be due to a point mutation in SS2 region. (2) The cDNA clone of TuNa I linked to RSV promotor gave us variable expression efficiency and we synthesized TuNa I mRNA from cDNA to inject ascidian neural blastomeres to clarify causes of the variability. (3) We found high efficiency of expression, when a part of 5'end of the coding region of thymidine kinase mRNA was linked to the head of the coding region of TuNa I mRNA,and concluded that TuNa I was useful for a reporter gene at least in ascidian embryos. (4) In order to discriminate exogenous expression of TuNa I from the endogenous one we have tried to altemate the TTx insensitive channels to the TTX sensitive ones by site-directed mutagenesis in the region of SS2.
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