Wan Yongjie, Deng Mingtian, Zhang Guomin, Ren Caifang, Zhang Hao, Zhang Yanli, Wang Lizhong, Wang Feng
Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing , 210095, China.
Cell Biol Int. 2016 Jan;40(1):74-82. doi: 10.1002/cbin.10540. Epub 2015 Sep 14.
Somatic cell nuclear transfer (SCNT) is a useful way to produce cloned animals. However, SCNT animals exhibit DNA methylation and genomic imprinting abnormalities. These abnormalities may be due to the faulty epigenetic reprogramming of donor cells. To investigate the consequence of SCNT on the genomic imprinting and global methylation in the donor cells, growth patterns and apoptosis of cloned goat fibroblast cells (CGFCs) at passage 7 were determined. Growth patterns in CGFCs were similar to the controls; however, the growth rate in log phase was lower and apoptosis in CGFCs were significantly higher (P < 0.01). In addition, quantitative expression analysis of three DNA methyltransferases (Dnmt) and two imprinted genes (H19, IGF2R) was conducted in CGFCs: Dnmt1 and Dnmt3b expression was significantly reduced (P < 0.01), and H19 expression was decreased sixfold (P < 0.01); however, the expression of Dnmt3a was unaltered and IGF2R expression was significantly increased (P < 0.05). Finally, we used bisulfite sequencing PCR to compare the DNA methylation patterns in differentially methylated regions (DMRs) of H19 and IGF2R. The DMRs of H19 (P < 0.01) and IGF2R (P < 0.01) were both highly methylated in CGFCs. These results indicate that the global genome might be hypomethylated. Moreover, there is an aberrant expression of imprinted genes and DMR methylation in CGFCs.
体细胞核移植（SCNT）是一种用于生产克隆动物的有效方法。然而，SCNT动物表现出DNA甲基化和基因组印记异常。这些异常可能是由于供体细胞的表观遗传重编程错误所致。为了研究SCNT对供体细胞基因组印记和整体甲基化的影响，我们测定了第7代克隆山羊成纤维细胞（CGFCs）的生长模式和凋亡情况。CGFCs的生长模式与对照相似；然而，对数期的生长速率较低，且CGFCs中的凋亡率显著更高（P < 0.01）。此外，我们对CGFCs中的三种DNA甲基转移酶（Dnmt）和两个印记基因（H19、IGF2R）进行了定量表达分析：Dnmt1和Dnmt3b的表达显著降低（P < 0.01），H19的表达下降了六倍（P < 0.01）；然而，Dnmt3a的表达未改变，IGF2R的表达显著增加（P < 0.05）。最后，我们使用亚硫酸氢盐测序PCR来比较H19和IGF2R的差异甲基化区域（DMRs）中的DNA甲基化模式。H19（P < 0.01）和IGF2R（P < 0.01）的DMRs在CGFCs中均高度甲基化。这些结果表明，整体基因组可能发生了低甲基化。此外，CGFCs中存在印记基因的异常表达和DMR甲基化。
