| 陈访访,贾怡昌.小鼠大脑抑制性神经元特异性多聚核糖体结合型mRNA的提取[J].,2017,17(11):2001-2006 |
| 小鼠大脑抑制性神经元特异性多聚核糖体结合型mRNA的提取 |
| Isolation of Inhibitory Neuron-specific Polyribosome-associated mRNA from Mouse Brain |
| 投稿时间：2016-10-08 修订日期：2016-10-30 |
| DOI：10.13241/j.cnki.pmb.2017.11.001 |
| 中文关键词: 小鼠 大脑 抑制性神经元特异性 多聚核糖体结合型 mRNA |
| 英文关键词: Mouse Brain Inhibitory neuron-specific Polyribosome-associated mRNA |
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| 中文摘要: |
| 摘要 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。 |
| 英文摘要: |
| ABSTRACT Objective: To establish a rapid and efficient method in extracting the Inhibitory Neuron-specific Polyribosome-associ- ated mRNA from Mouse Brain, provide materials for the analysising of inhibitory Neuron-specific expression spectrum from the mouse brain.. Methods: Based on the cre-loxp system, we crossed the RiboHA mouse to VGAT/PV-Cre mouse, to activate the expression of HA-tagged ribosomes. The genotype of the offspring was analyzed to obtain the Cre and HA co-expressed mouse. The expression of HA was detected by immunofluorescent staining. The HA-tagged polyribosomes were purified from the target cell population via immuno- precipitation. The mRNAs was isolated from the immunoprecipitated polyribosomes. The cell-type specificity of mRNAs was analyzed using quantitative RT-PCR (qRT-PCR). The quality of mRNAs and cDNAs was checked by using agarose gel electrophoresis and bioana- lyzer 2100. Results: The expression of HA-tagged polyribosomes was efficiently activated in the target cell populations. The polyribosome- associated mRNAs were enriched and isolated from the inhibitory neurons specifically. The quality of cell-type-specific polyribosome-as- sociated mRNAs and cDNAs was high enough for the high-throughput sequencing and translating profiling analysis. Conclusion: Through the Cre-loxp genetic labeling system combined with the immunoprecipitation, inhibitory neuron-specific polyribosome-associated mRNA transcripts have been isolated rapidly and efficiently from the mouse brain. The results lay the foundation for further analysis of the specific expres- sion of the inhibitory neurons in the brain of mice. |
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