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（由申请人提供）：中枢神经系统（CNS）神经元之间的连接映射是神经科学家的长期目标。对联系的知识将为我们了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学以及蛋白质的转移均已用于跟踪突触连接。还利用了它们在神经元之间传播的能力，也使用了神经性病毒。病毒示踪剂提供了更高吞吐量的承诺，并可以通过其基因传输能力扩大映射连接超出映射连接的测定曲目。我们最近开发了囊泡口腔炎病毒（VSV）作为透射性示踪剂。该病毒比先前开发的透射性病毒，狂犬病病毒（RABV）和伪比病毒（PRV）具有多个优点。 RABV是一种致命的病毒，它限制了其在实验室中的使用。 PRV具有大型且复杂的基因组，因此很难以直接的方式设计。 VSV具有相对简单的基因组，与RABV非常相似。它的特征非常好，因为它迅速增长到高质量，并且在实验室安全方面具有很长的记录。我们发现，VSV可以在小鼠，猴子和其他多种物种（包括鸟类，两栖动物和鱼类）中透射传递。此外，我们发现我们可以创建可以特异性逆转或特定前进的VSV向量。方向性完全取决于由病毒G基因编码的病毒包膜糖蛋白。我们建议创建一系列的透射病毒示踪剂，这些示踪剂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。我们将首先探索使用不同病毒G蛋白的VSV的使用，并将我们所学的知识应用于特定的逆行和逆行示踪剂的设计。我们希望扩大不仅可以与VSV一起使用的G蛋白的曲目，还可以与其他示踪剂一起使用。我们还建议使用已经为VSV开发的一些工具开发一种替代性无毒病毒作为示踪剂。最后，我们建议开发一个示踪剂，尽管基于病毒，但实际上不是病毒，而是一种将CRE重组酶跨突触移动以绘制连接神经元的载体。这些替代策略提供了相对于VSV，RABV或PRV降低毒性的希望，以及可以与小鼠工程线或其他模型生物结合使用的其他功能。