BDNF PROMOTER ELEMENTS IN THE DEVELOPING HYPOTHALAMUS

描述(改编自申请者的描述)：本报告的目标 提案有两个方面。第一个是对监管和作用的定性 下丘脑中的脑源性神经营养因子(BDNF)。第二个是 将首席调查员发展为独立研究员。 脑源性神经营养因子属于神经营养因子家族中的神经生长因子。 它的功能对神经系统的正常发育至关重要。更多 最近，人们认识到，在正常运作中发挥关键作用 成人的大脑。调查人员最近描述了一种 NT缺陷小鼠的有趣表型，其中单倍体缺乏BDNF 老鼠会变得肥胖或过度活跃。在转录水平上，这些 表型的特征是BDNF在细胞内的不同调节 下丘脑。他们假设表型的差异可能是由于 脑源性神经营养因子的转录调控BDNF有四个启动子，它们是 每个外显子都与它们自己的未翻译外显子以及第五个编码相关联 外显子。 本提案将评估这些外显子中的每一个的表达和功能 特别是下丘脑核团在体外和体内的应用 模特们。通过研究每个未翻译的BDNF外显子的表达， 研究人员可以确定哪些启动子元件是关键和特异的 到特定的下丘脑核团。在具体目标1中，他们将分析 以细胞特有的方式表达这些未翻译的外显子中的每一个 在下丘脑内。他们将通过利用Lacz记者来做到这一点 针对每个外显子的构建以及随后对这些外显子的转染 构建成器官型下丘脑培养物。《特定目标2》将针对 最有趣的启动子在体内的发育表达 具体目的1通过建立稳定转基因小鼠品系。 最后，具体目标3将解决最具体的体内功能 通过在小鼠胚胎干细胞中引入突变来获得这些启动子 研究人员已经发现的破坏基因的关键启动子元件 抄写。研究人员计划培育的小鼠只会 在需要靶向启动子元件的领域缺乏BDNF 将会发生转录。通过这种方式，他们可以在分子水平上确定 靶向启动子的功能。

DESCRIPTION (Adapted from applicant's description): The goals of this proposal are twofold. The first is to characterize the regulation and role of brain derived neurotrophic factor (BDNF) in the hypothalamus. The second is the development of the principal investigator as an independent researcher. BDNF belongs to the nerve growth factor (NGF) family of neurotrophins (NTs) and its function is critical for normal nervous system development. More recently, it has been appreciated to play key roles in the proper functioning of the adult brain. The investigators have recently characterized an interesting phenotype of NT deficient mice whereby haplo-insufficient BDNF mice become obese or hyperactive. At the transcriptional level these phenotypes are characterized by differential regulation of BDNF within the hypothalamus. They hypothesize that divergence in phenotype may be due to the transcriptional control of BDNF. There are four promoters for BDNF that are each associated with their own untranslated exon along with a fifth coding exon. This proposal will evaluate the expression and function of each of these exons in particular hypothalamic nuclei by the use of both in vitro and in vivo models. By studying the expression of each untranslated BDNF exon, the investigators can determine which promoter elements are critical and specific to particular hypothalamic nuclei. In Specific Aim 1 they will analyze the expression of each of these untranslated exons in a cell specific manner within the hypothalamus. They will do this by utilizing lacZ reporter constructs specific for each exon and subsequent transfection of these constructs into organotypic hypothalamic cultures. Specific Aim 2 will address the in vivo developmental expression of the most interesting promoters from Specific Aim 1 by establishing a line of stably transfected transgenic mice. Finally, Specific Aim 3 will address the in vivo function of the most specific of these promoters by introducing a mutation into mouse embryonic stem cells of key promoter elements which the investigators have shown disrupt gene transcription. The mice that the investigators propose to generate will only be deficient in BDNF in areas that need the targeted promoter elements for transcription to occur. In this manner they can determine at a molecular level the function of the targeted promoter.