Characterization of GnRH/GFP Transgenic Rats

GnRH/GFP 转基因大鼠的表征

• 批准号: 7239059
• 负责人: ROBERT C THOMPSON
• 金额: $ 2.64万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7239059
• 关键词: biological signal transduction; bioluminescence; electrodes; electrophysiology; gene expression; genetically modified animals; genotype; gonadotropin releasing factor; green fluorescent proteins; immunocytochemistry; laboratory rat; laser capture microdissection; microarray technology; neurons; polymerase chain reaction; reproduction; single cell analysis; transfection

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.

描述（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。在缺乏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（每脑约1000-1200个），并且分布在基底前脑和下丘脑，因此很难在电生理和分子水平上进行研究。为了解决这些限制，我们将生产一种转基因大鼠，其中增强的绿色荧光蛋白（eGFP）在大鼠GnRH启动子的控制下表达，该启动子先前被证明可以正确地将转基因表达靶向GnRH神经元。这种转基因大鼠的发展将使人们能够很容易地通过GFP的荧光特性来鉴定GnRH神经元，而无需进行重大的组织处理，重要的是，这种鉴定在活细胞（例如组织切片制备）中是可行的。由于在这个物种中有大量的行为学、神经内分泌学和神经解剖学的文献，大鼠是这类研究的理想对象。此外，大鼠是一种理想的动物模型，与生殖研究相关的一系列手术、药物和其他治疗程序已经建立起来。我们提供了设计的初步数据，我们的转基因的体外分析，并记录了13个转基因创始大鼠的存在（即在其基因组中含有我们的转基因）。在本应用中，我们计划使用联合免疫细胞化学和eGFP荧光技术验证eGFP在转基因大鼠中的表达仅限于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，我们希望我们的策略在大鼠中同样有效。一旦GFP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。这两项研究都将侧重于利用GFP指导细胞鉴定来评估单个GnRH神经元中的基因表达。这些策略将包括(1)激光捕获显微镜和(2)使用电生理记录电极进行单细胞分离。我们为此应用程序开发了两种方法，因为每种方法都有其特定的优点和缺点。这些基因表达研究最初将集中于鉴定已知在GnRH神经元中表达的受体（gpcr）和离子通道（亚基）rna（以及几种新型rna），这将证实并扩展我们对能够修饰GnRH分泌的信号类型的认识。综上所述，这些初步的表征研究将是验证我们的转基因动物的关键，然后再开始对GnRH神经元内的基因表达模式进行广泛的评估和电生理研究。