Transgenic Tethered Peptides

描述(由申请人提供)：我们的长期目标是开发工具来表征神经肽在完整行为动物体内的神经回路和非神经组织中的功能。我们已经开发了一种新的技术，在这种技术中，神经肽被转基因表达为嵌合融合蛋白，通过疏水锚点(t-肽)连接到质膜上。T-肽在表达T-肽的细胞中具有预期的特异性激活其同源G蛋白偶联受体(GPCRs)，但在不表达T-肽的邻近细胞的膜上不激活其GPCRs。因此，T-肽为转基因动物神经肽功能的细胞自主功能增益分析提供了遗传编码的工具。这些特定的目标旨在提供一个全基因组工具包，其中包括遗传编码的神经肽GPCRs的细胞自主药理特异性激活剂。由于这个工具包将基于UAS-GAL4二元表达系统，该系统将细胞和电路特异的启动子GAL4驱动基因从UAS转基因基因中分离出来，UAS转基因基因包含编码t-肽等效应物的cDNA，因此它将对对神经肽的细胞特异性生理和行为功能感兴趣的果蝇生物学家有直接的帮助。更广泛地说，T-肽文库的开发和验证也将提供宝贵的见解，使工具的产生能够用于解决活体哺乳动物神经肽在健康和疾病中的功能，包括在基因治疗应用中的潜在临床应用。 与公共健康相关：生物活性的分泌神经肽是许多发育、生理和行为过程的关键调节者：新陈代谢、寿命、痛感、昼夜节律、睡眠、性行为等。神经肽信号机制功能障碍导致的人类疾病--如发作性睡病、肥胖、成瘾、创伤后应激障碍、顽固性疼痛等--是发病率、死亡率和经济困难的主要来源。通过了解后生动物基因组中编码的大量神经肽在体内的正常功能，将有助于它们的改善。

DESCRIPTION (provided by applicant): Our long-term goal is the development of tools for characterizing the functions of neuropeptides in neural circuits and non-neuronal tissues in vivo in intact behaving animals. We have developed a new technology, where neuropeptides are transgenically expressed as chimeric fusion proteins tethered to the plasma membrane via hydrophobic anchors ("t-peptides"). T-peptides activate their cognate G-protein coupled receptors (GPCRs) with expected specificity in t-peptide-expressing cells, but without activating their GPCRs in the membranes of neighboring cells not expressing the t-peptide. T- peptides thus provide genetically encoded tools for the cell-autonomous gain-of-function analysis of neuropeptide function in transgenic animals. The Specific Aims are designed to provide a genome-wide toolkit of genetically encoded cell-autonomous pharmacologically specific activators of neuropeptide GPCRs. Because this toolkit will be based on the UAS-GAL4 binary expression system that separates cell- and circuit- specific promoter GAL4 driver transgenes from UAS transgenes containing cDNAs that encode effectors such as t-peptides, it will be of direct utility to Drosophila biologists interested in cell-specific physiological and behavioral functions of neuropeptides. More generally, the development and validation of t-peptide libraries will also provide invaluable insights enabling the generation of tools for use in addressing mammalian neuropeptide function in vivo in health and disease, including potential clinical utility in gene therapy applications. PUBLIC HEALTH RELEVANCE: Bioactive secreted neuropeptides are key regulators of many developmental, physiological, and behavioral processes: metabolism, lifespan, pain sensation, circadian rhythms, sleep, sexual behavior, etc. Human disorders caused by dysfunction of neuropeptide signaling mechanisms-such as narcolepsy, obesity, addiction, post- traumatic stress disorder, intractable pain, etc-are a major source of morbidity, mortality, and economic hardship. Their amelioration will be facilitated by understanding the normal in vivo functions served by the numerous neuropeptides encoded in metazoan genomes.