Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes
Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes
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DOI：
10.4161/epi.6.10.17202
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发表时间：
2011-10-01
期刊：
影响因子：
3.7
通讯作者：
Haaf, Thomas
中科院分区：
文献类型：
--
作者：
El Hajj, Nady;Trapphoff, Tom;Haaf, Thomas
为了检测与辅助生殖相关的罕见表观遗传效应，有必要监测一些生殖细胞和单个胚胎中发育重要基因的甲基化模式。亚硫酸氢盐处理会降解 DNA 并降低其复杂性，使得少量 DNA 的甲基化分析极具挑战性。在这里，我们描述了一种简单的方法，可以确定单个早期胚胎中多个基因的亲本特异性甲基化模式。亚硫酸氢盐处理 DNA 的有限稀释 (LD) 与单个 DNA 靶分子的独立多重 PCR 相结合，以避免扩增偏差。使用这种方法，我们比较了三组不同的单小鼠双细胞胚胎中三个印记基因（H19、Snrpn 和 Igf2r）和一个多能性相关基因（Oct4）的甲基化状态。与体内产生的对照相比，超排卵母细胞的标准体外受精和体外成熟卵母细胞的使用与随机单 CpG 甲基化错误和表观突变（等位基因甲基化错误）率的显着增加无关。同样，我们比较了从超排卵和非超排卵母细胞体内产生的个体小鼠 16 细胞胚胎中两个印记基因（H19 和 Snrpn）的甲基化模式，并且没有观察到主要的组间差异。使用牛卵母细胞和极体作为模型，我们证明 LD 甚至可以对单个细胞中的多个基因进行甲基化分析。
To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.