用于非人灵长类动物特定类型神经元的稀疏精细标记工具

Neuron is the unit of neural circuit. Definition of the neuronal morphology will facilitate to analyze the neural circuit and the neuronal function. Monkey as animal model has been used in the field of neuroscience research, which will be good for elucidate the human brain working mechanism. Currently, several viral tracer can infect neuron of monkey, however, they have some drawbacks, such as the low efficiency of protein expression, non-cell specific labeling, and so on. Therefore, these tools do not have ability of cell type-specific sparse labeling the neuron in non-human primates. In order to solve the problem and develop the tool for the analysis of neuronal morphology of monkey, we previously have developed a tool for non-cell specific labeling neuron of monkey. Here, we plan to take advantage of my research experience in virology and the recent advances in the relevant fields to develop new tool for cell type-specific sparse labeling tool for the neuron of the non-human primate..Semliki foreset virus (SFV) belongs to the family Togaviridae genus Alphavirus. The genome is a single-stranded, positive-sense RNA of about 12 kb in length, encoding four non-structural proteins and five structural proteins. The structural and nonstructural proteins are for virion assembly and viral replication, respectively. The reverse genetic systems of SFV, such as replicon and single round infection of virus-like particle, are broadly available. .In this study, dopamine neuron is selected as a model and the human tyrosine hydroxylase is selected as a cell-type specific cell marker. The human tyrosine hydroxylase gene promoter can induce expression of the reporter EGFP in dopamine neurons. Combination of the hybird of DNA virus, Cre-lox and EnvA-TVA system, we will use the methods of reverse genetics, viral recombinant, cell culture, virus injection, and fluorescence microscope to develop a tool for cell-type specific labeling of the monkey neuron. The tool will be used as helper virus in developing trans-synaptic tracer for monkey neural circuit, and the strategy will be served as a model for developing various tools for labeling cell-type specific neuron.

神经元是神经环路的组成单元，稀疏精细描绘其形态对于了解神经元的功能和神经环路的构成具有重要作用。猴作为非人灵长类动物已被用于神经科学研究，其研究结果对于推导人脑工作机制具有重要意义。目前感染猴脑神经元的工具病毒存在表达蛋白能力弱、没有细胞特异性等问题而无法实现特定神经元的稀疏精细标记。申请人前期以高效表达外源蛋白的西门利克森林病毒为对象建立了非特异稀疏精细标记猕猴脑神经元的工具。本项目拟以多巴胺神经元为范式，开展1）明确酪氨酸羟化酶基因启动子的特异性序列；2）优化西门利克森林病毒的表达体系；3）结合病毒杂合、Cre-lox和EnvA-TVA等方面的研究，采用病毒重组和在体标记等技术，建立用于猴脑多巴胺神经元的稀疏精细标记工具。若项目得以实施，不仅为研究猴脑多巴胺神经元提供有效工具，而且为制备其它类型神经元的稀疏精细标记工具提供参考范式，同时也可作为猴脑特定环路示踪的辅助工具病毒。

神经细胞是神经环路的组成单元，稀疏精细描绘其形态对于了解神经细胞的功能和神经环路的构成具有重要作用。申请人前期以高效表达外源蛋白的西门利克森林病毒为对象建立了非特异稀疏精细标记猕猴脑神经元的工具，在此基础上进行突变，建立了可快速稀疏精细标记神经细胞的工具系统。通过一系列的摸索，明确了病毒进入神经细胞的位点为胞体，并明确了其在体内的最佳感染时间和取样成像时间。为开展神经细胞的形态标记提供了快速有效可用的工具。