Wei Yanchang, Zhu Jiang, Huan Yanjun, Liu Zhongfeng, Yang Cairong, Zhang Xinmiao, Mu Yanshuang, Xia Ping, Liu Zhouhua
College of Life Science, Northeast Agricultural University , Harbin, People's Republic of China.
Cell Reprogram. 2010 Apr;12(2):213-22. doi: 10.1089/cell.2009.0090.
Unlike embryos derived from fertilization, most cloned embryos die during postimplantation development, and those that survive to term are frequently defective. Many of the observed defects involve placenta. Abnormal placentation has been described in several cloned species. Imprinted genes are important regulators of placenta growth, and may be subjected to faulty reprogramming during somatic cell nuclear transfer. We aimed to determine the expression levels and methylation patterns of imprinted genes in placentas of live cloned piglets and dead ones. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the expression of all four imprinted genes (IGF2, H19, PEG3, and GRB10) was significantly reduced in placentas of dead clones compared with placentas of live cloned piglets and controls (p < 0.05). In contrast, both live and dead cloned piglets exhibited steady-state mRNA levels for these genes within the control range (p > 0.05). Transcript levels for these genes in live clones rarely differed from those of controls in both piglets and placentas. Examination of the methylation status of DMR2 of IGF2 and CTCF3 of H19 genes revealed that both genes exhibited significant high methylation levels in placentas of dead clones compared with placentas of live clones and controls. In contrast, both genes showed a normal differential methylation pattern in live cloned piglets and their placentas compared with controls. Importantly, dead cloned piglets also showed a normal pattern. Our results suggest that abnormal expression of imprinted genes in placenta may contribute to the development failure in pig somatic cell nuclear transfer (SCNT), which may be caused by abnormal methylation patterns in differentially methylated regions (DMRs) of imprinted genes as a result of incomplete reprogramming during SCNT.
与受精产生的胚胎不同，大多数克隆胚胎在植入后发育过程中死亡，而那些存活至足月的克隆胚胎也常常存在缺陷。许多观察到的缺陷都涉及胎盘。在多个克隆物种中都描述过胎盘形成异常的情况。印记基因是胎盘生长的重要调节因子，在体细胞核移植过程中可能会经历错误的重编程。我们旨在确定存活的克隆仔猪和死亡的克隆仔猪胎盘中印迹基因的表达水平和甲基化模式。定量实时逆转录聚合酶链反应（RT-PCR）分析表明，与存活的克隆仔猪胎盘和对照相比，死亡克隆仔猪胎盘中所有四个印记基因（IGF2、H19、PEG3和GRB10）的表达均显著降低（p < 0.05）。相比之下，存活和死亡的克隆仔猪中这些基因的稳态mRNA水平均在对照范围内（p > 0.05）。在仔猪和胎盘中，存活克隆仔猪中这些基因的转录水平与对照相比很少有差异。对IGF2的DMR2和H19基因的CTCF3甲基化状态的检测显示，与存活克隆仔猪胎盘和对照相比，死亡克隆仔猪胎盘中这两个基因均表现出显著的高甲基化水平。相比之下，与对照相比，存活克隆仔猪及其胎盘中这两个基因均显示出正常的差异甲基化模式。重要的是，死亡克隆仔猪也表现出正常模式。我们的结果表明，胎盘中印记基因的异常表达可能导致猪体细胞核移植（SCNT）发育失败，这可能是由于SCNT过程中重编程不完全导致印记基因差异甲基化区域（DMRs）的甲基化模式异常所致。
