Development of novel transsynaptic tracers for use in the central nervous system

开发用于中枢神经系统的新型突触示踪剂

• 批准号: 9268809
• 负责人: CONSTANCE L CEPKO
• 金额: $ 36.5万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9268809
• 关键词: Amphibia; Anatomy; Animal Model; Area; Biological Assay; Birds; Communication; Complex; Development; Disease; Dyes; Electron Microscopy; Engineering; Enterobacteria phage P1 Cre recombinase; Fishes; G-substrate; GTP-Binding Proteins; Gene Transfer; Genes; Genome; Glycoproteins; Goals; Health; Hippocampus (Brain); Infection; Interneurons; Knowledge; Label; Laboratories; Learning; Maps; Methods; Monkeys; Mus; Nervous System Physiology; Nervous system structure; Neuraxis; Neurons; Organism; Physiological; Preparation; Proteins; Rabies virus; Reagent; Retina; Safety; Series; Synapses; Testing; Toxic effect; Tracer; Transgenic Mice; Variant; Vesicular stomatitis Indiana virus; Viral; Viral Vector; Virus; Virus-like particle; base; cell type; design; in vivo; mad itch virus; nervous system disorder; neurotropic virus; novel; particle; public health relevance; receptor; tool; transmission process; vector

DESCRIPTION (provided by applicant): The mapping of connections among central nervous system (CNS) neurons is a longstanding goal of neuroscientists. Knowledge of the connections will inform our understanding of the function of the nervous system, in both health and disease. Dye labeling, physiological recordings, anatomy at the level of electron microscopy, and the transfer of proteins have all been used to track synaptic connections. Neurotropic viruses have also been used, taking advantage of their ability to spread among neurons. Viral tracers offer the promise of higher throughput, and can expand the repertoire of assays beyond mapping connections, via their gene transfer capabilities. We have recently developed vesicular stomatitis virus (VSV) as a transsynaptic tracer. This virus has several advantages over the previously developed transsynaptic viruses, rabies virus (RABV) and pseudorabies virus (PRV). RABV is a lethal virus, which limits its use in the laboratory. PRV has a large and complex genome, making it difficult to engineer in a straightforward manner. VSV has a relatively simple genome, very similar to that of RABV. It is very well characterized as it rapidly grows to high titr and has a long track record for safety in the laboratory. We found that VSV can transmit transsynaptically in mice, monkeys, and multiple other species, including birds, amphibians, and fish. Moreover, we found that we could create VSV vectors that could transmit specifically retrogradely or specifically anterogradely. The directionality was entirely dependent upon the viral envelope glycoprotein, encoded by a viral G gene. We propose to create a series of transsynaptic viral tracers that will be useful to neuroscientists working in different organisms and different areas of the nervous system. We will first explore the use of VSV with different viral G proteins, applying what we learn to the design of specific anterograde and retrograde tracers. We hope to expand the repertoire of G proteins that can be used not only with VSV, but with other tracers as well. We also propose to develop an alternative non-toxic virus for use as a tracer, using some of the tools that we have already developed for VSV. Finally, we propose to develop a tracer that, although based upon a virus, is actually not a virus, but a vehicle to move Cre recombinase across synapses to map connected neurons. These alternative strategies offer the promise of reduced toxicity, relative to VSV, RABV, or PRV, as well as other features that can be combined with engineered lines of mice, or other model organisms.

描述(申请人提供)：绘制中枢神经系统(CNS)神经元之间的联系图是神经科学家的长期目标。对这些联系的了解将有助于我们理解神经系统在健康和疾病中的功能。染料标记、生理记录、电子显微镜水平的解剖以及蛋白质的转移都被用来追踪突触连接。嗜神经性病毒也被使用，利用它们在神经元中传播的能力。病毒示踪剂提供了更高吞吐量的前景，并可以通过它们的基因转移能力，将分析的范围扩展到映射连接之外。我们最近开发了水泡性口炎病毒(VSV)作为跨突触示踪剂。这种病毒比以前开发的跨突触病毒、狂犬病病毒(RABV)和伪狂犬病病毒(PRV)有几个优点。RABV是一种致命病毒，这限制了它在实验室中的使用。PRV基因组庞大而复杂，很难以简单的方式进行工程设计。VSV的基因组相对简单，与RABV非常相似。它具有很好的特性，因为它迅速生长到高滴度，并在实验室中有长期的安全记录。我们发现VSV可以在小鼠、猴子和包括鸟类、两栖动物和鱼类在内的多种其他物种中跨突触传播。此外，我们发现我们可以创建VSV载体，这种载体可以特定地逆行传播或特定地顺行传播。方向性完全取决于病毒包膜糖蛋白，由病毒G基因编码。我们建议创造一系列跨突触的病毒示踪剂，这将对在不同生物体和神经系统不同区域工作的神经科学家有用。我们将首先探索VSV与不同的病毒G蛋白的用途，将我们所学到的应用于特定的顺行和逆行示踪剂的设计。我们希望扩大G蛋白的谱系，不仅可以与VSV一起使用，也可以与其他示踪剂一起使用。我们还建议使用我们已经为VSV开发的一些工具来开发一种替代的无毒病毒作为示踪剂使用。最后，我们建议开发一种示踪剂，虽然基于病毒，但实际上不是病毒，而是一种载体，可以在突触之间移动Cre重组酶，以映射连接的神经元。与VSV、RABV或PRV相比，这些替代策略提供了降低毒性的前景，以及可以与小鼠工程化品系或其他模式生物相结合的其他特征。