Effect of DNA Methylation onIgf-2r Expression in the Development of Cloned Cattle

Abstract：Epigenetic reprogramming has a crucial role in establishing nuclear totipotency in normal development and in cloned animals. In the current study, the method of Real-time fluorescent quantitative PCR (FQ-PCR) was applied to detect Igf2r mRNA in Madin–Darby bovine kidney（MDBK ）cells after being treated with 5’-azacytidine (5’-aza, a DNA methyltransferase inhibitor). And then we used the method of Bisulfite DNA Sequencing to detect DNA methylation status of Igf-2r DMR 2（DNA differentially methylated region，DMR）and 3’UTR（3’-untranslated region，UTR）in several tissues, such as brain, liver, lung, and heart in normal and cloned cattle. Results suggested that Igf2r mRNA were up-regulated in MDBK cells after being treated with 5’-aza. DNA methylation at DMR 2 significantly varied in normal cattle tissues but without significant variation at Igf-2r 3¢-UTR. DNA methylation at Igf-2r DMR2 in cloned cattle was markedly altered compared with a normal fetus, while it was similar to a normal fetus at Igf-2r 3¢-UTR. Based on the results, it is suggested that DNA methylation inhibitor down-regulated the expression of Igf-2r in MDBK cells. In normal cattle, patterns of DNA methylation at Igf-2r DMR2 was variable in different tissues, suggesting that the mechanism of gene imprinting was differently regulated in these tissues. Results also showed that in cloned cattle, Igf-2r DMR2 DNA methylation was disrupted while the non-imprinting control region (3'-UTR) was not. It suggested that disruption of the gene imprinting control region was likely to result in the abnormalities of cloned cattle.