Disruption of imprinting in cloned mouse fetuses from embryonic stem cells
Disruption of imprinting in cloned mouse fetuses from embryonic stem cells
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DOI：
10.1530/rep.0.1260549
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发表时间：
2003-10-01
期刊：
影响因子：
3.8
通讯作者：
Kono, T
中科院分区：
文献类型：
--
作者：
Ogawa, H;Ono, Y;Kono, T
克隆小鼠通常表现出发育异常，如胎儿和胎盘过度生长。为了探讨印迹基因的表观遗传修饰是否与胚胎和胎盘的过度生长和胎盘肥大有关，采用实时荧光定量聚合酶链式反应技术分析了8个印记基因(H19、lgf2、lgf2r、Air、PEG1/Mest、PEG3、Nuronatin(NNAT)和NDN)及其交替转录产物lgf2(PO)在胚胎干细胞克隆胚胎和胎盘交配后9.5、12.5和17.5d的定量表达。此外，通过亚硫酸氢盐测序法检测了9.5天胎儿和胎盘中H19和lgf2r差异甲基化区域的甲基化模式。结果表明，克隆胎儿在配对后12.5天和17.5天，胎盘中H19基因的表达在配对后17.5天明显降低，而lgf2在配对后9.5和12.5天和配对后17.5天在胎盘中也受到抑制。相反，在克隆胎盘中，在交配后12.5天，胎盘特异性转录变体PO的转录增加了4倍以上。第9.5天正常发育的胎儿只在H19差异甲基化区(DMR)发现了高甲基化的等位基因，而在Igf2r DMR2中既有高甲基化的等位基因，也有低甲基化的等位基因。这些结果表明，某些印记基因的不适当重编程影响克隆胚胎的发育，特别是PO lgf2转录异常可能导致克隆胎儿和胎盘的过度生长。
Cloned mice typically display abnormal development, such as overgrowth of fetuses and placentae. Quantitative expression analysis of eight imprinted genes (H19, lgf2, lgf2r, Air, Peg1/Mest, Peg3, Nuronatin (Nnat) and Ndn) and an alternate transcript of lgf2 (PO) in embryonic stem cloned fetuses and placentae at days 9.5, 12.5 and 17.5 after mating was carried out by real time PCR to investigate whether epigenetic modification of imprinted genes is responsible for overgrowth of the fetus and placental hypertrophy. In addition, the methylation pattern through the bisulphite sequencing method in differentially methylated regions of H19 and lgf2r was examined in day 9.5 fetuses and placentae. The results showed clearly that the expression of H19 gene decreased in cloned fetuses at days 12.5 and 17.5 after mating and in placentae at day 17.5 after mating, and lgf2 was also repressed in fetuses at days 9.5 and 12.5 after mating and in placentae at day 17.5 after mating. In contrast, the transcription of PO, which is a placental-specific transcript variant of lgf2, increased at more than four times the control in cloned placenta at day 12.5 after mating. Day 9.5 fetuses that have developed normally revealed only hypermethylated alleles in the H19 differently methylated region (DMR), and both hyper- and hypomethylated alleles in the Igf2r DMR2. These results show that inappropriate reprogramming in some imprinted genes affects the development of cloned embryos, and that aberrant PO lgf2 transcription in particular may cause the overgrowth of cloned fetuses and placentae.