Ogawa H, Ono Y, Shimozawa N, Sotomaru Y, Katsuzawa Y, Hiura H, Ito M, Kono T
Department of BioScience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
Reproduction. 2003 Oct;126(4):549-57. doi: 10.1530/rep.0.1260549.
Cloned mice typically display abnormal development, such as overgrowth of fetuses and placentae. Quantitative expression analysis of eight imprinted genes (H19, Igf2, Igf2r, Air, Peg1/Mest, Peg3, Nuronatin (Nnat) and Ndn) and an alternate transcript of Igf2 (P0) 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 Igf2r 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 Igf2 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 P0, which is a placental-specific transcript variant of Igf2, 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 P0 Igf2 transcription in particular may cause the overgrowth of cloned fetuses and placentae.
克隆小鼠通常表现出发育异常，如胎儿和胎盘过度生长。通过实时PCR对交配后第9.5天、12.5天和17.5天的胚胎干细胞克隆胎儿及胎盘中8个印记基因（H19、Igf2、Igf2r、Air、Peg1/Mest、Peg3、神经调节蛋白（Nnat）和Ndn）以及Igf2的一个可变转录本（P0）进行定量表达分析，以研究印记基因的表观遗传修饰是否与胎儿过度生长和胎盘肥大有关。此外，采用亚硫酸氢盐测序法检测了第9.5天胎儿及胎盘中H19和Igf2r差异甲基化区域的甲基化模式。结果清楚地表明，交配后第12.5天和17.5天的克隆胎儿以及交配后第17.5天的胎盘中H19基因表达下降，交配后第9.5天和12.5天的胎儿以及交配后第17.5天的胎盘中Igf2也受到抑制。相反，P0（Igf2的胎盘特异性转录变体）在交配后第12.5天的克隆胎盘中转录增加，是对照组的四倍多。正常发育的第9.5天胎儿在H19差异甲基化区域（DMR）仅显示高甲基化等位基因，在Igf2r DMR2中则同时显示高甲基化和低甲基化等位基因。这些结果表明，某些印记基因的重编程不当会影响克隆胚胎的发育，特别是异常的P0 Igf2转录可能导致克隆胎儿和胎盘过度生长。
