Department of Animal Sciences, Institute of Food and Agriculture Sciences, University of Florida, Gainesville, FL, USA.
Syst Biol Reprod Med. 2010 Feb;56(1):96-105. doi: 10.3109/19396360903331148.
Characterization of DNA methylation is one assessment of chromatin remodeling in early embryos. Unfortunately, evaluation at specific loci is hindered by their small cell numbers. Our objective was to determine if bisulfite sequencing could be optimized for preimplantation embryos, comparing conversion times, primer design, and DNA amplification methods. Methylation at three loci, SATI, OCT4, and IGF2, was investigated in bovine in vitro produced (IVP) embryos, somatic cells, and no template controls. Bisulfite treatment for 15-16 h gave higher quality DNA than treatment for 18 h. Three step primer design improved bisulfite primer specificity, yielding more PCR product than primers previously reported. Following optimization, methylation data were obtained from as few as 4 cell equivalents. Finally, DNA amplification efficiencies were evaluated using miniprep, TempliPhi, or 96-well glycerol stocks with automated TempliPhi. While TempliPhi was better than standard minipreps, the 96-well format proved most efficient. Preliminary methylation profiles of bovine IVP 2-cell, 8-cell, blastocyst stage embryos and somatic cells were 25, 10, 22, and 74% for SATI and 88, 88, 79, and 88% for OCT4, respectively, suggesting that SATI is demethylated during early embryonic reprogramming, while OCT4 remains hypermethylated. IGF2 methylation was 84, 28, and 84% for bovine IVP 8-cell, blastocyst stage embryos and somatic cells; blastocyst stage embryos exhibited more variability, ranging from 0 to 80%. This new assay will enhance assessment of chromatin remodeling in embryos, and be especially useful for evaluating those produced by assisted reproductive technologies.
DNA 甲基化的特征是早期胚胎染色质重塑的一种评估方法。不幸的是，由于其细胞数量较少，对特定基因座的评估受到阻碍。我们的目标是确定亚硫酸氢盐测序是否可以针对胚胎进行优化，比较转化时间、引物设计和 DNA 扩增方法。在牛体外生产 (IVP) 胚胎、体细胞和无模板对照中，研究了三个基因座 SATI、OCT4 和 IGF2 的甲基化。15-16 小时的亚硫酸氢盐处理比 18 小时的处理产生更高质量的 DNA。三步引物设计提高了亚硫酸氢盐引物的特异性，产生的 PCR 产物比以前报道的引物多。经过优化，从 4 个细胞当量即可获得甲基化数据。最后，使用 miniprep、TempliPhi 或 96 孔甘油储存液（带有自动化 TempliPhi）评估 DNA 扩增效率。虽然 TempliPhi 优于标准 miniprep，但 96 孔格式证明效率最高。牛 IVP 2 细胞、8 细胞、囊胚阶段胚胎和体细胞的初步甲基化谱分别为 SATI 的 25%、10%、22%和 74%，OCT4 的 88%、88%、79%和 88%，表明 SATI 在早期胚胎重编程过程中去甲基化，而 OCT4 仍然高度甲基化。牛 IVP 8 细胞、囊胚阶段胚胎和体细胞的 IGF2 甲基化分别为 84%、28%和 84%；囊胚阶段胚胎的变异性更大，范围为 0 至 80%。这种新的检测方法将增强对胚胎染色质重塑的评估，特别是对辅助生殖技术产生的胚胎的评估非常有用。
