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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.
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