Establishment And Application Of Single Cell Sequencing Based On Tracing Specific Neural Circuit By Rabies Virus

Rabies virus is a lethal neurotropic microorganism.Because of its high lethality and the lack of good treatment methods,it poses a great threat to the health of humans and related host animals,and it has become a very important pathogen in Etiology.On the other hand,due to its special tropism for neurons and its ability to survive and replicate in neurons,the rabies virus has been developed into a powerful tool that can be used to trace and label specific neural circuits.It has some natural advantages as opposed to the traditional neural circuits tracing tools and possess great application value and potential in related neuroscience research.The brain,as the most complex structure and function organ in animal body,has become the hottest and most challenging field in life sciences and medical research.The in-depth research and exploration of it will provide theoretical significance and guidance for understanding the brain's operating mechanism and prevention of related brain diseases.The most important step in revealing the mysteries of the brain is the analysis of the structure and function of a large number of intricate neural circuits.Therefore,based on the above related technologies and scientific backgrounds,the main purpose of this project is using rabies virus to trace and label specific neural circuits for depicting the structural basis of the corresponding neural circuits,and combining single cell sequencing technology to performing transcriptome analysis of the neurons on the neural circuit at the single cell resolution for revealing the functional basis of the corresponding neural circuit.So we can realize the analysis of the structure and function of the specific neural circuit,and further explore the regulation mechanism of the neural circuit,providing a certain data base and research ideas for how to change when outside pathogens invade.The research content of this study is mainly divided into the following contents: 1.Purification and application of Tn5Tn5 is a transposase from E.coli,it has been developed as an enzyme tool for fragmentation and adapters ligation in next generation sequencing libraries construction with its ability to transposition in vitro and DNA fragmentation.It has a very wide application in single cell sequencing research.In this study,we firstly performed expression,purification and assembly of Tn5.Secondly we did the activity assay of Tn5,and verified its ability to DNA fragmentation and adapter ligation.Finally we applied it to the Smart-seq2 method for single-cell transcriptome sequencing successfully,which saved the cost of library construction and extended the application of special functions.2.Establishment of Smart-seq2 technology for the culture cells and the neurons of specific neural circuitSmart-seq2 technology is the most commonly used single-cell RNA-seq technology currently.We firstly applied it to the culture cells by constantly trying and fumbling and accomplished the transcriptome amplification from single cells.Then we applied it to the specific neural circuit neurons combining with specific neural circuit neuron labeling and separation technology using rabies virus and accomplished the libraries construction and sequencing from single neurons,realizing the exploration and function of the specific neural circuit structure of interest.3.The tracing and function preliminary exploration of PVN-LHA neural circuitPVN is a nerve nuclei in the hypothalamus that regulates the body's autonomy.The PVN-LHA neural circuit has a direct influence on the regulation of the body's diet.The in-depth study can understand the mechanism of brain's regulation to dietary behavior and response to pathogen invasion.In this study,we used the GFP-recombined rabies virus to label the PVN-LHA circuit and then performed a single-cell transcriptome analysis of the neurons labeled in the PVN.We revealed the genes expression files of these neurons and identified the expression of type and function related genes in neurons,providing a certain foundation for further research.In summary,this study combined the rabies virus tracer technique for specific neural circuits and single-cell RNA-seq technology to achieve the establishment and application of single cell transcriptome analysis technology for neurons on specific neural circuits,and then researched the PVN-LHA neural circuit using this method,achieving preliminary identification of the type and function of the neurons in the PVN of this neural circuit and providing a certain data base for the further study of this neural circuit.Furthermore the combination of these two technologies provides a technical foundation and research ideas for the related neuroscience fields.