High Resolution Imaging of DNA Methylation Dynamics using a Zebrafish Reporter.
High Resolution Imaging of DNA Methylation Dynamics using a Zebrafish Reporter.
复制标题
使用斑马鱼报告基因对 DNA 甲基化动态进行高分辨率成像。
DOI：
10.1038/s41598-017-05648-8
复制
发表时间：
2017-07-14
影响因子：
4.6
通讯作者：
Wang X
中科院分区：
文献类型：
--
作者：
Zhang R;Liu L;Yao Y;Fei F;Wang F;Yang Q;Gui Y;Wang X
作为主要的表观遗传修饰之一，DNA甲基化在胚胎发育、细胞谱系定型和病理过程中不断受到调节。为了便于实时观察DNA甲基化，我们通过敲入由bactin 2启动子驱动的mCherry融合的甲基-CpG结合结构域（MBD）探针来产生DNA甲基化的转基因斑马鱼报告基因（zebraRDM）。探针与异染色质共定位，其强度与早期胚胎亚细胞分辨率下的5 mC免疫染色呈正相关。生化分析表明，具有较强荧光的细胞保持较高水平的DNA甲基化，并在囊胚期的延时成像显示，DNA甲基化水平在有丝分裂过程中短暂加强。通过将zebraRDM与其他荧光转基因系杂交，我们证明了报告者可以在视觉上区分心脏等器官中的不同细胞谱系。因此，我们的zebraRDM报告基因是一种方便而强大的工具，可用于高分辨率研究活体动物中的甲基化动态。
As one of the major epigenetic modifications, DNA methylation is constantly regulated during embryonic development, cell lineage commitment, and pathological processes. To facilitate real-time observation of DNA methylation, we generated a transgenic zebrafish reporter of DNA methylation (zebraRDM) via knockin of an mCherry-fused methyl-CpG binding domain (MBD) probe driven by the bactin2 promoter. The probe colocalized with heterochromatin, and its intensity was positively correlated with 5 mC immunostaining at a subcellular resolution in early embryos. Biochemical assays indicated that cells with stronger fluorescence maintained a higher level of DNA methylation, and time-lapse imaging at the blastula stage showed that the level of DNA methylation was transiently strengthened during mitosis. By crossing zebraRDM with other fluorescent transgenic lines, we demonstrate that the reporter can visually distinguish different cell lineages in organs like the heart. Our zebraRDM reporter therefore serves as a convenient and powerful tool for high-resolution investigation of methylation dynamics in live animals.
登录
查看更多内容
影响因子：
64.5
作者：
Goessling W;North TE;Loewer S;Lord AM;Lee S;Stoick-Cooper CL;Weidinger G;Puder M;Daley GQ;Moon RT;Zon LI
通讯作者：
Zon LI
影响因子：
14.9
作者：
Jørgensen HF;Adie K;Chaubert P;Bird AP
通讯作者：
Bird AP
影响因子：
4.6
作者：
Hameed UF;Lim J;Zhang Q;Wasik MA;Yang D;Swaminathan K
通讯作者：
Swaminathan K
影响因子：
14.9
作者：
Calvanese V;Fernández AF;Urdinguio RG;Suárez-Alvarez B;Mangas C;Pérez-García V;Bueno C;Montes R;Ramos-Mejía V;Martínez-Camblor P;Ferrero C;Assenov Y;Bock C;Menendez P;Carrera AC;Lopez-Larrea C;Fraga MF
通讯作者：
Fraga MF
影响因子：
5.8
作者：
Kamstra, Jorke H.;Alestrom, Peter;Legler, Juliette
通讯作者：
Legler, Juliette