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目的:建立能够高效、快速地从小鼠大脑提取抑制性神经元特异性多聚核糖体结合的mRNA的方法,为进行小鼠大脑抑制性神经元特异性翻译表达谱分析提供材料。方法:依据Cre-loxp系统,将RiboHA标签小鼠与抑制性神经元特异性VGAT-Cre/PV-Cre小鼠杂交,启动抑制性神经元中核糖体上HA标签的表达。后代小鼠进行基因型鉴定,获得同时表达Cre和HA的小鼠。利用免疫荧光染色检测HA的表达。通过免疫共沉淀从目标细胞群体中获得HA标记的多聚核糖体。提取多聚核糖体结合的mRNA。荧光定量PCR法检测所得mRNA的细胞类型特异性。利用琼脂糖凝胶电泳及bioanalyzer 2100检测所得mRNA及cDNA的质量。结果:HA标记的多聚核糖体在目标细胞群体中能够被高效启动表达。抑制性神经元特异性多聚核糖体结合的mRNA能够被特异性地富集提取。所获细胞类型特异性的多聚核糖体结合的mRNA及cDNA的质量足以用来进行高通量测序及翻译表达谱的分析。结论:利用Cre-loxp遗传系统标记,结合蛋白免疫共沉淀实验,建立了从小鼠大脑抑制性神经元中高效特异地获得多聚核糖体结合的mRNA的方法,为进一步进行小鼠大脑抑制性神经元特异性翻译表达谱的分析奠定了基础。
OBJECTIVE: To establish a method for efficient and rapid extraction of inhibitory neuron specific ribosome-binding mRNA from the mouse brain and to provide material for the inhibition of mouse brain-specific neuronal-specific translational profiling. Methods: According to Cre-loxp system, RiboHA-tagged mice were crossed with inhibitory neuron-specific VGAT-Cre / PV-Cre mice to initiate HA tag expression on ribosomes in inhibitory neurons. Progeny mice were genotyped to obtain mice that express both Cre and HA. The expression of HA was detected by immunofluorescence staining. HA-tagged polysomes were obtained from the target cell population by co-immunoprecipitation. Ribosome-binding mRNA was extracted. Fluorescence quantitative PCR was used to determine the cell type specificity of the resulting mRNA. The quality of the obtained mRNA and cDNA was examined by agarose gel electrophoresis and bioanalyzer 2100. Results: The HA-tagged polysomes can be efficiently primed in the target cell population. Inhibitor-specific neuronal-specific polysome-bound mRNAs can be specifically and exogenously extracted. The quality of the obtained cell type-specific polysome-bound mRNA and cDNA is sufficient for high-throughput sequencing and analysis of translational profiling. Conclusion: The method of Cre-loxp genetic system labeling and protein co-immunoprecipitation experiments was established to efficiently and specifically obtain ribosome-binding mRNA from mouse brain inhibitory neurons. In order to further study the inhibitory effects of mouse inhibitory nerve Meta-specific translational profiling laid the foundation for the analysis.