Characterization of GnRH/GFP Transgenic Rats

性状（由申请人提供）：促性腺激素释放激素（GnRH）在生殖的中枢调节中起关键作用。在没有GnRH的情况下，任何已知的哺乳动物都不可能繁殖。由于GnRH神经元数量很少（每个脑约1000 - 1200个）且分散于基底前脑和下丘脑，因此很难在电生理和分子水平上进行研究。为了解决这些局限性，我们将产生一种转基因大鼠，其中增强型绿色荧光蛋白（eGFP）在大鼠GnRH启动子的控制下表达，该启动子先前显示出将转基因表达正确靶向GnRH神经元。这种转基因大鼠的发展将允许人们通过GFP的荧光特性容易地识别GnRH神经元，而无需主要的组织处理，重要的是，这种识别在活细胞中是可行的（例如组织切片制备）。大鼠是这种研究的理想动物，因为在这一物种中有大量的行为学、神经内分泌学和神经解剖学文献。此外，大鼠是一种理想的动物模型，其一系列与生殖研究相关的外科手术、药理学和其他治疗程序已充分确立。我们目前的设计，我们的转基因的体外分析的初步数据和文件的存在13转基因创始人大鼠（即在其基因组中含有我们的转基因）。在本申请中，我们计划使用联合免疫细胞化学和eGFP荧光来验证eGFP的表达仅限于该转基因大鼠的GnRH神经元。鉴于三个独立的研究小组已经使用GnRH启动子成功地将eGFP，DGal或荧光素酶转基因靶向小鼠的GnRH神经元，我们预计我们的策略在大鼠中同样有效。一旦GFP的表达被证明是GnRH特异性的，我们计划进行两种类型的分子研究。这两项研究都将集中在使用GFP指导细胞鉴定的单个GnRH神经元中基因表达的评价上。这些策略将涉及（1）激光捕获显微镜和（2）使用电生理记录电极的单细胞分离。 我们已经为这个应用开发了两种方法，因为每种方法都有特定的优点和缺点。这些基因表达研究最初将重点关注已知在GnRH神经元内表达的受体（GPCR）和离子通道（亚基）RNA（以及几种新型RNA）的鉴定，这将证实和扩展我们对能够改变的信号类型的了解。GnRH分泌。总之，这些初步的表征研究将是关键，以验证我们的转基因动物开始广泛的评估GnRH神经元内的基因表达模式和电生理学研究。

DESCRIPTION (provided by applicant): Gonadotropin-releasing hormone (GnRH) plays a critical role in the central regulation of reproduction. In the absence of GnRH, reproduction is simply not possible in any known mammal. Because GnRH neurons are few in number (about 1000-1200 per brain) and scattered throughout the basal forebrain and hypothalamus, they are very difficult to study at the electrophysiological and molecular levels. To address these limitations, we will produce a transgenic rat in which the enhanced Green Fluorescent Protein (eGFP) is expressed under the control of the rat GnRH promoter, a promoter previously shown to correctly target transgene expression to GnRH neurons. The development of this transgenic rat will allow one to easily identify GnRH neurons by the fluorescent properties of GFP without major tissue treatments and importantly, such identifications are feasible in living cells (e.g. tissue slice preparations). The rat is ideal for such studies due to the vast behavioral, neuroendocrinological and neuroanatomical literature in this species. Further, the rat is an ideal animal model for which a series of surgical, pharmacological and additional treatment procedures relevant to the study of reproduction are well established. We present preliminary data on the design, in vitro analysis of our transgene and document the existence of 13 transgenic founder rats (i.e. contain our transgene in their genome). In this application, we plan to verify that the expression of eGFP is limited to GnRH neurons in this transgenic rat using combined immunocytochemistry and eGFP fluorescence. Given that three separate research groups have used the GnRH promoter to successfully target eGFP, DGal or luciferase transgenes to GnRH neurons in mice, we expect our strategy in rats to be equally effective. Once GFP expression is documented and shown to be GnRH specific, we plan to perform two types of molecular studies. Both of these studies will focus on the evaluation of gene expression in single GnRH neurons using GFP to guide cell identification. These strategies will involve (1) laser capture microscopy and (2) single cell isolation using electrophysiological recording electrodes. We have developed both methods for this application as each has particular strengths and weaknesses. These gene expression studies will initially focus on the identification of receptors (GPCRs) and ion channel (subunit) RNAs known to be expressed within GnRH neurons (as well as several novel RNAs) that will confirm and expand our knowledge of the types of signals capable of modifying GnRH secretion. Taken together these initial characterization studies will be key to validating our transgenic animal prior to embarking on broad evaluations of gene expression patterns within GnRH neurons and electrophysiological studies.