小鼠α-synuclein基因内含子1甲基化定量检测方法的建立及不同脑区甲基化水平的比较研究

Establishment of a method to measure the methylation level of mouse α-synuclein intron-1 and comparison of its methylation at different brain regions

目的建立一种定量检测小鼠α-突触核蛋白基因(α-syn)内含子1甲基化水平的方法,分析不同脑区该区域甲基化水平的差异。方法使用MSPPrimer确定小鼠α-syn内含子1的CpG岛区域。使用PyroMark assay design 2.0针对该CpG岛设计焦磷酸扩增及测序引物。采用焦磷酸测序法分析成年小鼠海马、皮质、纹状体及中脑四个脑区该区域甲基化水平。结果小鼠α-syn内含子1存在CpG岛。成功优化定量该CpG岛甲基化水平的焦磷酸测序方法。焦磷酸测序表明各脑区该区域甲基化均低于2%。结论建立了定量检测小鼠α-syn基因内含子1CpG岛甲基化水平的方法。该CpG岛甲基化对于成年小鼠各脑区中α-syn的表达不起主要调控作用。

Objective To establish a quantitative method to examine the methylation level of CpG island located in the intron 1 of mouse α-synuclein, and analyze the difference of methylation in various brain regions .Methods CpG island in the intron 1 of mouse α-synuclein was identified using an online software MSPprimer .Pyrosequencing assay was designed using PyroMark assay design 2.0.DNA methylation was quantified in mouse hippocampus , cortex, striatum and midbrain using pyrosequencing assay .Results There was a CpG island in the intron 1 of mouse α-synuclein. Pyrosequencing assay measuring methylation level of this CpG island was optimized .It indicated that all of the brain regions were hypomethylated with a methylation rate of 2%or less.Conclusion DNA methylation does not play a major role in the transcription regulation of mouse α-synuclein in the brain .