Dual PRV Transsynaptic Labeling: EGFP & mRFP1 Reporters

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追踪技术。最近，双重病毒transneuronal标记已被引入采用重组株的PRV Bartha工程表达不同的报告蛋白。双重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重组株表达由不同启动子驱动的不同报告基因，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株作为双重跨突触示踪剂的潜力。我们已经开发了两种PRV Bartha的同基因重组株（即，PRV152和PRV614）的差异仅在于它们表达的荧光报告蛋白。PRV 152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。PRV 152被广泛使用，并得到了很好的表征。PRV 614表达一种新的单体红色荧光蛋白（mRFP1），由CMV启动子驱动，也插入在gG基因的中间。它是最近才建成的，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具有巨大的实用性，这些示踪剂可以同时可视化，而无需额外的组织处理。在本申请中，我们建议将新开发的PRV 614表征为跨突触逆行病毒示踪剂，其可与PRV 152组合使用以进一步定义大脑中的神经元回路。PRV 614感染和逆行转运的动力学将使用PRV通过支配眼睛的自主神经回路的逆行转运在体内确定。将在体内和体外测定当一种PRV比另一种晚到达时，两种PRV的同基因株感染相同神经元的能力。Transneuronal逆行双PRV标记有可能成为一个强大的除了神经解剖学工具的神经回路的调查，PRV 614将消除许多陷阱与目前使用的双PRV重组体。

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.