The Methylation Status Of IGF2/H19 And IGF2R Gene In Different Tissues Of Newborn Cloned Pig

Porcine somatic cell cloning technology plays an important role in animal husbandry production, medical research, therapeutic cloning and the protection of rare and endangered animals. However, the low efficiency of cloning and developmental abnormalities of cloned animals had seriously hampered the development and application of the technology. The incomplete reprogramming of donor cells was the most important reason for the low efficiency of cloning and developmental abnormalities of cloned animals. Normal expression of imprinted genes are essential during the growth and development of cloned animal embryos, and its abnormal DNA methylation will cause abnormal expression of imprinted genes or unexpressed, lead to fet al loss and the abnormalities and so on.We have obtained two Fengjing pigs by somatic cell cloning, and one was died when at birth. In order to make sure the imprinting model of the imprinted gene, and whether the DNA methylation reprogramming of imprinted genes is efficient. We analyzed the DNA methylation status of IGF2/H19 and IGF2R differentially methylated region in heart, liver, spleen, lung and kidney of cloned pig, and these organs of normal pig by bisulfite sequencing analysis. Also the methylation status of IGF2/H19 and IGF2R in the fetal fibroblast cells are detected, so as to explore the reprogramming mechanism of donor cell in nuclear transfer, which may provide a theoretical basis for improving the efficiency of porcine somatic cell cloning. And the main results are as follows:(1) The methylation profiles of IGF2/H19 gene DMR1 and DMR3 in heart, liver, spleen, lung and kidney of cloned pigs and normal pig were different, but the difference was not significant (P> 0.05);(2) The methylation level of IGF2/H19 DMR2 in cloned pig lung and kidney of was higher than the control pig, especially in the lungs, it was hypermethylated, and was significantly higher than normal pigs (95.20% vs 46.80%,P<0.01), while in other tissues, the difference was not significant(P>0.05);(3) The IGF2R DMR2 showed hypermethylation in liver of the cloned pig, and was significantly higher than the control (80.00%vs39.41% P<0.05); while in lung of the dead cloned pig, the IGF2R DMR was hypomethylated, and was ignificantly lower than the control (14.71% vs 66.47% P<0.01), in others tissues the differences were insignificant (P>0.05);(4) The methylation level of IGF2/H19 DMR2 in fetal fibroblast cells were not significant different with that in heart, liver, spleen, kidney cloned pig, but was significant different with that in lung of cloned pig(55.60% vs 95.20%, P< 0.01);(5) The methylation level of IGF2R DMR2 in fetal fibroblast cells were not significant different with that in heart, spleen, kidney cloned pig(P> 0.05), but was significant different with that in liver and lung of cloned pig(48.24% vs 80.00%, P< 0.05; 48.24% vs 14.71%, P< 0.01).Results showed that the methylation status of IGF2/H19 DMR2 in cloned pig lung were abnormal, also the methylation status of IGF2R DMR in cloned pig lung and liver were abnormal. These results demonstrated that the incomplete DNA methylation reprogramming had occurred in the donor cell during the process of nuclear transfer.