Dual PRV Transsynaptic Labeling: EGFP & mRFP1 Recepters

双 PRV 突触标记：EGFP

• 批准号: 6766727
• 负责人: GARY Edward PICKARD
• 金额: $ 11.88万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766727
• 关键词: cytomegalovirus; electrophysiology; green fluorescent proteins; hamsters; laboratory rat; neuroanatomy; neuronal transport; neurophysiology; recombinant virus; spinal ganglion; suid alphaherpesvirus 1; synapses; tissue /cell culture; voltage /patch clamp

DESCRIPTION (provided by applicant): The transsynaptic retrograde transport of the Bartha strain of pseudorabies virus (PRV Bartha) has become an important neuroanatomical tract-tracing technique for the characterization of neuronal circuits in the central nervous system. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful technique for defining interactions between parallel neural circuits in the brain. However, the current use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods, limits the potential of these recombinant PRV Bartha strains as dual transsynaptic tracers. We have developed two isogenic recombinant strains of PRV Bartha (i.e., PRV152 and PRV614) differing only in the fluorescent reporter protein they express. PRV152 expresses the enhanced green fluorescent protein (EGFP), is driven by the human cytomeglovirus (CMV) promoter, and is inserted in the middle of the gG gene in the middle of the viral genome. PRV152 is in wide use and is well characterized. PRV614 expresses a novel monomeric red fluorescent protein (mRFP1) driven by the CMV promoter also inserted in the middle of the gG gene. It has only recently been constructed and is uncharacterized. The availability of two viral transneuronal tracers expressing different fluorescent reporters that can be visualized concurrently without additional tissue processing has enormous utility. In this application, we propose to characterize the newly developed PRV614 as a transsynaptic retrograde viral tracer that can be used in combination with PRV152 to further define neuronal circuits in the brain. The kinetics of PRV614 infection and retrograde transport will be determined in vivo using the retrograde transport of PRV through autonomic circuits innervating the eye. The ability of two isogenic strains of PRV to infect the same neuron when one PRV arrives later than the other will be determined both in vivo and in vitro. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; PRV 614 will eliminate many of the pitfalls associated with the currently used dual PRV recombinants.

描述（由申请人提供）：伪狂犬病病毒 Bartha 株（PRV Bartha）的跨突触逆行转运已成为表征中枢神经系统神经元回路的重要神经解剖学纤维束追踪技术。最近，通过使用 PRV Bartha 重组菌株引入了双重病毒跨神经元标记，该菌株经过改造可表达不同的报告蛋白。双病毒突触追踪有潜力成为一种极其强大的技术，用于定义大脑中并行神经回路之间的相互作用。然而，目前使用表达不同报告基因的PRV重组毒株，这些报告基因由不同的启动子驱动，插入病毒基因组的不同区域，并通过不同的方法检测，限制了这些重组PRV Bartha毒株作为双跨突触示踪剂的潜力。我们开发了两种 PRV Bartha 的同基因重组株（即 PRV152 和 PRV614），其区别仅在于它们表达的荧光报告蛋白。 PRV152表达增强型绿色荧光蛋白（EGFP），由人巨细胞病毒（CMV）启动子驱动，插入病毒基因组中部的gG基因中部。 PRV152 应用广泛且特性良好。 PRV614 表达一种由同样插入 gG 基因中部的 CMV 启动子驱动的新型单体红色荧光蛋白 (mRFP1)。它最近才建成并且没有特征。表达不同荧光报告基因的两种病毒跨神经元示踪剂的可用性可以同时可视化，无需额外的组织处理，具有巨大的实用性。在此应用中，我们建议将新开发的 PRV614 描述为一种跨突触逆行病毒示踪剂，可与 PRV152 结合使用，以进一步定义大脑中的神经元回路。 PRV614感染和逆行转运的动力学将利用PRV通过支配眼睛的自主神经回路的逆行转运在体内测定。当一种PRV比另一种晚到达时，两种PRV同基因株感染同一神经元的能力将在体内和体外测定。跨神经元逆行双 PRV 标记有可能成为神经元回路研究的神经解剖工具的有力补充； PRV 614 将消除与当前使用的双 PRV 重组体相关的许多缺陷。