小鼠外周组织时钟基因启动子区甲基化分析

Promoter methylation analysis of clock genes in mice peripheral tissues

目的应用一种简单快速经济的方法检测8个主要时钟基因PER1、PER2、CRY1、CRY2、CLOCK、NPAS2、BMAL1和BMAL2启动子区甲基化状态,检测小鼠外周组织肝、心、肾、胸腺、睾丸时钟基因启动子区甲基化状态。方法用偏重亚硫酸氢钠和对苯二酚对基因组DNA进行修饰。修饰后的DNA为模板,两套不同的引物对:甲基化特异性引物对和非甲基化特异性引物对扩增小鼠肝、心、肾、胸腺、睾丸组织时钟基因启动子区。PCR产物进行电泳和测序。结果扩增产物与预期片段大小相符合。PCR产物经过直接测序进一步证实小鼠外周组织时钟基因启动子区均为非甲基化状态。结论建立了一种检测小鼠时钟基因启动子区甲基化的新方法,借此证明成年小鼠5个外周组织8个时钟基因启动子区均呈非甲基化状态。

Objective Using a simple,rapid and inexpensive method to determine the methylation status of eight key clock genes,PER1,PER2,CRY1,CRY2,CLOCK,NPAS2,BMAL1 and BMAL2.To examine the methylation of clock promoters in five peripheral tissues.Methods Genome DNA was deaminated by Sodium metabisulfite solution and hydroquinone.The two sets of PCR primers for unmenthylated and methylated DNA,respectively,were used to amplify the promoter of clock genes.Polymerase chain reaction（PCR） products were then loaded and electrophoresed on 3% agarose gels.The PCR products for each reaction were sequenced.Results Specific amplicons with correct size were amplified.PCR products were also confirmed by sequencing and indicated that all of the clock promoters are not methylated.Conclusions All of the eight clock promoters are not methylated in the five adult mice peripheral tissues.