SCNT牛胚胎中PWS/AS基因簇母源印记控制区甲基化模式研究

摘要

In order to study the methylation pattern of clone embryos,early clone embryos at five developmental stages (gamophase, S phase,2-cell,8-cell and morula)were selected to study the methylation pattern of PWS/AS imprinting cluster by bisulfite sequencing PCR (BSP). The results of sequencing analysis indicated that demethylation pattern of genome was affected by the early methylation reconstruction of SCNT embryos. There were two CPG positions that were respectively the first and the fifth CPG position located at 258 bp and 1693 bp region on upstream of SNRPN transcriptional start.%为揭示牛核移植胚胎(SCNT)异常甲基化模式,选择配子期、S期、2-cell、8-cell和桑葚胚5个发育阶段的早期牛核移植胚胎作为试验组,以相应阶段的体外受精胚胎(IVF)作为对照,对核移植胚胎中呈母源印记的PWS/AS基因簇ICR区进行BSP测序,分析了SCNT和IVF胚胎不同发育时期SNRPN启动子区域甲基化水平的总体变化模式,并对该区域内的5个甲基化位点分别进行比较,筛选敏感位点.结果表明:SCNT胚胎PWS-IC甲基化水平,各期均无显著性差异(P>0.05),一直维持在较高水平;SCNT胚胎二细胞期PWS-IC甲基化水平极显著低于同期的IVF胚胎(P<0.01);该区域第1个和第5个甲基化位点是去甲基化抑制机制的易感位点.因此,PWS-IC区域甲基化模式改变,导致了SCNT胚胎母源DNA的高甲基化,推测与SCNT胚胎发生过早甲基化重建相关.研究结果可为提高SCNT胚胎克隆成功效率提供理论借鉴.