| Project/Area Number |
10670071
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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 |
Environmental physiology (including Physical medicine and Nutritional physiology)
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| Research Institution | Nippon Medical School |
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Principal Investigator |
KATO Masakatsu Nippon Medical School, Department of Physiology, Associate Professor, 医学部, 助教授 (90143239)
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| Co-Investigator(Kenkyū-buntansha) |
SAKUMA Yasuo Nippon Medical School, Department of Physiology , Professor, 医学部, 教授 (70094307)
NISHIHARA Masugi Tokyo University, Graduate School, Faculty of Agriculture and Life Science, Professor, 大学院・農学生命科学研究科, 教授 (90145673)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1998: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | GnRH neuron / GFP / transgenic / トランスジュニック |
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| Research Abstract |
GnRH neurons in the medial preoptic area and the hypothalamus play an important role in the central control of reproductive functions, such as onset of puberty and female ovulation cycle. However, physiological characterization of GnRH neurons has been done very little, because identification of GnRH neurons in the physiological experiment is very difficult. To overcome this difficulty, we decided to tag GnRH neurons with green fluorescent protein (GFP) by generating transgenic rats.
Transgene was constructed with a 3.14 kb promoter of rat GnRH gene, 0.64 kb intron of rabbit globin gene and 0.72 kb EGFP gene. The transgene was microinjected into fertilized eggs of the rat. The manipulated eggs were transferred into the pseudopregnant recipients. Integration of the transgene was screened by PCR and Southern blot analysis of tail DNA.Out of 66 pups born, 6 were positive to the transgene, among which 4 showed normal reproductive function. In all these 4 lines, EGFP were specifically expressed in GnRH neurons. We have started a primary culture of GnRH neurons from these lines for electrophysiological experiment. We are also preparing organotypic culture of GnRH neurons for the analysis of synaptic organization.
In conclusion, we succeeded to generate the transgenic rats in which EGFP is specifically expressed by GnRH neurons, although we encountered several difficulties at the early stage of this project.
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