Tagging and functional analysis of hypothalamic peptidergic neurons

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发挥着重要作用。然而，由于生理学实验中对GnRH神经元的鉴定非常困难，对GnRH神经元的生理特性研究很少。为了克服这一困难，我们决定通过产生转基因大鼠来标记GnRH神经元。转基因大鼠GnRH基因启动子为3.14 kb，兔珠蛋白基因内含子为0.64 kb，EGFP基因为0.72 kb。将转基因基因显微注射到大鼠受精卵中。将处理过的卵子转移到假孕受者体内。经聚合酶链式反应和尾DNA Southern杂交鉴定，66只仔猪中有6只转基因阳性，其中4只繁殖功能正常。在这4个细胞系中，GnRH神经元均有EGFP的特异表达。我们已经开始对这些细胞系的GnRH神经元进行原代培养，以进行电生理实验。我们还准备对GnRH神经元进行器官型培养，以分析突触组织。综上所述，我们成功地产生了GnRH神经元特异性表达EGFP的转基因大鼠，尽管我们在该项目的早期阶段遇到了一些困难。

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.