Dual PRV Transsynaptic Labeling: EGFP & mRFP1 Reporters

描述(申请人提供)：伪狂犬病病毒Bartha株(PRV Bartha)的跨突触逆行运输已成为表征中枢神经系统神经回路的一种重要的神经解剖学追踪技术。最近，通过使用重组PRV Bartha株来表达不同的报告蛋白，引入了双重病毒跨神经元标记。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，目前使用的表达不同报告基因的重组PRV株由不同的启动子驱动，插入病毒基因组的不同区域，并通过不同的方法进行检测，限制了这些重组PRV Bartha株作为双重跨突触示踪剂的潜力。我们开发了两株PRV Bartha的同源重组株(即PRV152和PRV614)，只是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，插入病毒基因组中部GG基因中间。PRV152应用广泛，具有很好的特性。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，也插入在GG基因的中间。它是最近才建造的，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显示，而不需要额外的组织处理，具有巨大的实用价值。在这一应用中，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结合使用来进一步定义大脑中的神经元电路。PRV614感染和逆行转运的动力学将在体内利用PRV通过支配眼睛的自主神经回路的逆行转运来确定。当一种PRV到达另一种时，两种相同基因的PRV株感染同一神经元的能力将在体内和体外确定。跨神经元逆行双PRV标记有可能成为研究神经元电路的神经解剖学工具的有力补充；PRV 614将消除与当前使用的双PRV重组体相关的许多缺陷。

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.