Optimization of the Fluorescent Protein Expression Level Based on Pseudorabies Virus Bartha Strain for Neural Circuit Tracing.

Fan Jia,Pei Lv,Fuqiang Xu,Xiaoqin Xu,Hongjun Mei,Huan Miao,Sijue Tao,Li Li,Xiangwei Shi

doi:10.3389/fnana.2019.00063

Abstract

Mapping the neural circuits facilitates understanding the brain’s working mechanism. Pseudorabies virus (PRV; Bartha stain) as a tracer can infect neurons and retrogradely transport in neural circuits. To illuminate the network, tracers expressing reporter genes at a high level are needed. In this study, we optimized the expression level of reporter genes and constructed two new retrograde trans-multisynaptic tracers PRV531 and PRV724, which separately express more robust green and red fluorescent proteins than the existing retrograde tracers PRV152 and PRV614. PRV531 and PRV724 can be used for mapping the neural circuit of the central nervous system (CNS) and the peripheral nervous system (PNS). Overall, our work adds two valuable tracers to the toolbox for mapping neural circuits.

Highlights

One of the core tasks of modern neuroscience is to map neural circuits of the central nervous system (CNS) and the peripheral nervous system (PNS)
PRV152 as a trans-multisynaptic tool has been constructed by inserting the enhanced green fluorescent protein (EGFP) expression cassette into gG from the Pseudorabies virus (PRV) Bartha strain (Smith et al, 2000), which has been widely used in depicting the neural network of the CNS and PNS (Collins et al, 1999; Zhang et al, 2003; Kc et al, 2006; Kirby et al, 2010; Gonzalez-Joekes and Schreurs, 2012; Chen et al, 2013; Yao et al, 2018; Jin et al, 2019)
The brain section was treated with EGFP antibody and more EGFP neurons were detected (Figure 1), which indicates that PRV152 has the drawback of low EGFP expression level

Summary

Introduction

One of the core tasks of modern neuroscience is to map neural circuits of the central nervous system (CNS) and the peripheral nervous system (PNS). To depict the neural network, several tracers have been developed, such as Vesicular stomatitis virus and Herpes simplex virus type 1 (strain 129) can map the output information of a target brain region, while Rabies virus and Pseudorabies virus (PRV; Bartha strain) have the ability to reveal the input neural circuit Among these tracers, the PRV Bartha strain is the only retrograde trans-multisynaptic tool, and it is indispensable to determining the input neural circuit of the CNS and PNS (Collins et al, 1999; Zhang et al, 2003; Kc et al, 2006; Zhao, 2008; Kirby et al, 2010; Gonzalez-Joekes and Schreurs, 2012; Chen et al, 2013; Griffiths, 2015; Yao et al, 2018; Jin et al, 2019). The gG gene is nonessential for PRV replication, and a previous study engineered a retrograde trans-synaptic tracer by inserting a fragment of cytomegalovirus’s immediate early promoter, a single copy of reporter and a simian virus 40 poly(A) signal into

Methods

Results

Conclusion