Tagging and functional analysis of hypothalamic peptidergic neurons

内侧视前区和下丘脑的GnRH神经元在生殖功能的中枢控制中起重要作用，如青春期的开始和女性排卵周期。然而，GnRH神经元的生理表征做得很少，因为在生理实验中识别GnRH神经元非常困难。为了克服这一困难，我们采用绿色荧光蛋白（GFP）标记GnRH神经元的方法，构建了含有大鼠GnRH基因3.14 kb启动子、兔珠蛋白基因0.64 kb内含子和0.72 kb EGFP基因的转基因大鼠GnRH神经元。将转基因显微注射到大鼠的受精卵中。将经处理的卵子转移到假孕受体中。用PCR和Southern杂交技术检测转基因鼠尾DNA的整合情况，在66只出生的幼仔中，有6只转基因鼠尾DNA阳性，其中4只具有正常的生殖功能。在这4个细胞系中，EGFP均在GnRH神经元中特异性表达。我们从这些细胞系开始了GnRH神经元的原代培养，用于电生理实验。总之，我们成功地建立了GnRH神经元特异性表达EGFP的转基因大鼠，尽管我们在这个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.