Dual PRV Transsynaptic Labeling: EGFP & mRFP1 Reporters

描述（由申请人提供）：伪狂犬病毒Bartha株（PRV Bartha）的经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通道追踪技术。近年来，利用重组PRV Bartha表达不同的报告蛋白，引入了双病毒跨神经元标记。双病毒跨突触追踪有可能成为一种非常强大的技术，用于定义大脑中平行神经回路之间的相互作用。然而，目前使用的PRV重组菌株表达由不同启动子驱动的不同报告子，插入到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菌株作为双跨突触示踪剂的潜力。我们开发了两种PRV Bartha等基因重组菌株（即PRV152和PRV614），它们表达的荧光报告蛋白不同。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，插入病毒基因组中间gG基因的中间位置。PRV152具有广泛的用途和良好的特性。PRV614表达一种新的单体红色荧光蛋白（mRFP1），该蛋白由CMV启动子驱动，该启动子也插入gG基因的中间。它是最近才建成的，没有特征。两种表达不同荧光报告的病毒跨神经元示踪剂的可用性可以同时可视化，而无需额外的组织处理，具有巨大的实用性。在这项应用中，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，可以与PRV152结合使用，进一步定义大脑中的神经元回路。PRV614感染和逆行运输的动力学将在体内通过支配眼睛的自主神经回路逆行运输来确定。当一个PRV晚于另一个到达时，两个等基因PRV株感染同一神经元的能力将在体内和体外被确定。跨神经元逆行双PRV标记有可能成为神经解剖学工具的强大补充，用于研究神经元回路；PRV 614将消除与目前使用的双PRV重组病毒相关的许多缺陷。

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.