Construction Of VSV-SAD Chimeric Glycoprotein Pseudotyped Rabies Virus And Its Tunable Complementary AAV System

Decoding the structure and function of neural circuit is central in neuroscience research. "Science Watch" of 2012 selected Connectomes as one of the top 6important projects in the coming years. The elucidation of neural circuit depends on the neural circuit tracers. The neurotropic virus have been extensively and successfully engineered to explore neural circuit, as they contain the following features: Firstly, They can spread through synapses; Secondly, we can control the spreading direction to realize anterograde or retrograde tracing; Thirdly, the tracing signal will not fade out due to the replication of viruses in cells; Finally, we can engineer viruses by inserting interested genes into the genome of viruses.Although neurotropic virus such as rabies virus, pseudorabies virus and vesicular stomatitis virus have been widely used to unravel the input pathway of neural circuit,there are still some demerits in the toxicity, trans-synapse, neurotropism and safety of viruses.In our current research, we are aiming to construct a novel circuit tracing system for tracking the alteration of input pathway. We carried out our study based on a neurotropic virus, Rabies virus(RV). Through the insertion of fluorescent protein gene into the genome of a glycoprotein-deleted variant of RV SAD strain and the controllable expression of glycoprotein, we can get a less toxic but more safer recombinant viruses tracing system with specific marker. The following studies were carried out:1. Construction and identification of slow fluorescent timerWe constructed the gene of a slow fluorescent timer and inserted it into the genome of Adeno-associated virus to generate the recombinant virus AAV-CMV-Slow FT.After 12 hours infection of cells, the blue fluorescent gradually reached its maximum value and decreased later. When the red fluorescent reached its maximum value after24 hours, the blue fluorescent faded away.2. Construction and identification of double controllable gene expression system containing Tet-on and DD regulatory elementsWe construct a double regulatable system with Tet-on elements and Destabilizing Domain(DD) to control the expression of target gene. After trying different assembling mode of two Tet-on elements with DD, we finally got the best effective system by combing the TREtight responsible element of Tet-on with DD into the same AAV genome, while the rt TA of Tet-on were inserted into RV gemone. The double regulatory system is fast responsible, highly efficient and reversible. Moreover,it can reduce the leaking expression of target gene due to the single regulatory element. The system is identified both in vitro and in vivo.3. Constructing the AAV helper virus with controllable G protein under double regulatory systemTo control the expression of G protein, we constructed the G gene with double regulatory system. Due to the different virulence of the distinct RV strains including Shanxi, Anhui, SAD and N2 C strains, we construct various AAV helper viruses with glycoprotein from these strains. So we can compare the toxicity and neurotropism of different glycoprotein to finally screen the mildest but highly neurotropic glycoprotein for RV tracing.4. Construction, rescue and in vivo identificaiton of rabies virus with slow fluorescent timerThrough reverse genetic manipulation, we delete the glycoprotein(G protein) gene and insert a slow fluorescent timer(slow FT) gene into the genome of rabies virus at the same time. The recombinant virus has been rescued successfully. We inject the virus into the hippocampus of mouse by stereotaxic instrument to identify the colour change of slow fluorescent timer.5. Pseudotype rabies virus with VSV- SAD chemeric glycoproteinIn order to label the neurons of injection site to track the input pathway, we packaged the pseudotyped RV with a VSV-SAD chimeric glycoprotein. The chimeric G will be provided by B7BVSV-SAD G cell line which the genome is inserted BFP and VSV-SAD G genes but deleted GFP gene. The cell line is under construction.Trans-synaptic tracing is mainly based on the neurotropic virus. It’s the most efficient method to decode the structure of neural circuit and it’s also a rapid developing field in neuroscience research. Our novel virus tracing system will provide effective tracing tools for the study of neural circuit’s alteration and in vivo infection of Rabies virus.