Division of Epigenomics and Development, Medical Institute of Bioregulation, and Epigenome Network Research Center, Kyushu University, Fukuoka 812-8582, Japan, The Cancer Institute, Tangshan People's Hospital, Hebei 063001, China, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, F-67404 Illkirch, Cité Universitaire de Strasbourg, France, Department of Epigenetics Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi 409-3898, Japan, Division of Medical Genetics, Saitama Children's Medical Center, Saitama 339-8551, Japan and Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 113-8656, Japan.
Nucleic Acids Res. 2013 Oct;41(19):e186. doi: 10.1093/nar/gkt766. Epub 2013 Aug 28.
Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.
甲基化特异性荧光原位杂交（MeFISH）技术被开发出来，用于在单个细胞中特异性重复序列的 DNA 甲基化状态进行微观可视化。MeFISH 基于 5-甲基胞嘧啶和靶 DNA 中胞嘧啶对锇和含双吡啶的核酸（ICON）形成链间复合物的反应性差异。用荧光标记的 ICON 探针杂交的细胞核和染色体与用于交联的锇一起孵育。变性后，在野生型而非 DNA 甲基转移酶三重敲除（阴性对照）小鼠胚胎干细胞中，卫星重复序列特异性保留荧光信号。此外，使用 MeFISH，我们成功地在免疫缺陷、着丝粒不稳定和面部异常综合征患者的细胞中检测到卫星重复序列的低甲基化，以及在男性生殖细胞中检测到 5-羟甲基化卫星重复序列，后者基于抗 5-甲基胞嘧啶抗体染色曾被认为是非甲基化的。MeFISH 将非常适合在表观遗传学研究和医学诊断中的广泛应用。
