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Tagging and functional analysis of hypothalamic peptidergic neurons

下丘脑肽能神经元的标记和功能分析

基本信息

批准号：
10670071
负责人：
KATO Masakatsu
金额：
$ 2.11万
依托单位：
Nippon Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670071/
关键词：
GnRH neuron GFP transgenic トランスジュニック

项目摘要

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.

内侧视前区和下丘脑的GnRH神经元在青春期开始和女性排卵周期等生殖功能的中枢控制中起着重要作用。然而，由于GnRH神经元在生理实验中的鉴定非常困难，因此对GnRH神经元的生理表征研究很少。为了克服这一困难，我们决定通过产生转基因大鼠，用绿色荧光蛋白（GFP）标记GnRH神经元。用大鼠GnRH基因启动子3.14 kb、兔珠蛋白基因内含子0.64 kb和EGFP基因0.72 kb构建转基因。将转基因微注射到大鼠受精卵中。经过处理的卵子被移植到假怀孕的受者体内。通过PCR和尾部DNA的Southern blot分析筛选转基因的整合。66只出生幼崽中，转基因阳性6只，生殖功能正常4只。在这4个细胞系中，EGFP均在GnRH神经元中特异性表达。我们已经开始从这些细胞系中培养GnRH神经元进行电生理实验。我们也在准备GnRH神经元的器官型培养，用于分析突触组织。总之，尽管我们在项目初期遇到了一些困难，但我们成功地培育出了EGFP被GnRH神经元特异性表达的转基因大鼠。