2003 Fiscal Year Final Research Report Summary
Analysis of tissue specific expression of ChAT mRNA splice variants by in situ FRET method.
| Project/Area Number |
14580727
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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 |
Nerve anatomy/Neuropathology
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| Research Institution | Shiga University of Medical Sciences |
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Principal Investigator |
AIMI Yoshinari Molecular Neuroscience Research Center, Assistant Professor, 分子神経学研究センター, 助手 (20231756)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUO Akinori Molecular Neuroscience Research Center, Assistant Professor, 分子神経学研究センター, 助手 (20324585)
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| Project Period (FY) |
2002 – 2004
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| Keywords | pChAT / choline acetyltransferase / splice variant / FRET / histochemistry / PCR |
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| Research Abstract |
The purpose of this study was to establish a new "in situ fluorescence resonance energy transfer (FRET)" method for separate detection of pChAT mRNA variants in tissue sections. To select target tissues for this study, we first evaluated expression of pChAT in various tissues by pChAT immunohistochemistry and Westernblot. Dorsal root ganglion (DRG) and enteric nervous system were selected as targets. Then, we tried to detect pChAT mRNA by "in vitro" FRET. Full-length pChAT cDNA, which is inserted into a plasmid, was used as a positive control. A quantitative PCR method successfully demonstrated an occurrence of FRET specific to pChAT cDNA. Since a pair of FRET probe that is designed for pChAT did not emit any FRET fluorescence against cChAT, this method seemed to be suitable for separate detection of splice variants. The method, furthermore, was shown to be useful for quantitative analysis of pChAT mRNA. We tried to develop an "in situ FRET" method for pChAT next. DRG sections were incubated with a pair of FRET probes for pChAT. The sections were observed under a confocal laser microscope. Images composed with fluorescence around 640 nm (due to FRET) were similar to those obtained by pChAT immunohistochemistry. A spectrofluometrical analysis of the "in situ FRET" image clearly indicated that FRET signals were specific to pChAT. However, the quality of the images was relatively poor, therefore, we have continued experiments to improve the method. Among those experiments, we tried an extra experiment that combined the "in situ FRET" and "in situ PCR". To intensify the FRET signals, DRG sections were subjected to "in situ PCR" and simultaneous hybridization with FRET probes in one step procedure. Although this "in situ PCR-FRET" method is still preliminary, FRET signals were also detected as in "in situ FRET" method.
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