中文摘要：

目前认为克隆效率低的主要原因是供体核的不完全重编程导致发育过程中一些重要的基因异常表达。运用DNA甲基化转移酶抑制剂5＇-脱氧胞苷（5＇-azacytidine，5＇-aza）处理MDBK细胞（牛肾上皮细胞），并通过实时荧光定量PCR方法对lgf-2r基因的表达进行了定量分析；在此基础上，应用亚硫酸盐甲基化测序法检测正常牛及克隆牛脑、肺、心、肝组织lgf-2r印迹调控区DMR2（DNA differentially methylated region，DMR）及非印迹调控区3＇-UTR（3＇-untranslated region，UTR）的DNA甲基化水平。研究发现，5＇-aza处理MDBK细胞后，lgf-2r基因的表达上调。正常牛各组织中lgf-2rDMR2区的DNA甲基化程度差异较大，3＇-UTR区较稳定；与正常牛相比，克隆牛DMR2区的甲基化程度变化较大，3＇-UTR区无显著性变化。结果表明，DNA甲基化修饰影响lgf-2r基因的表达。正常牛不同组织中lgf-2r基因DMR2区的DNA甲基化程度不同，提示lgf-2r基因的印迹调控方式在不同组织中可能不同。克隆牛发育过程中，调控lgf-2r基因印迹的DMR2表观结构被明显改变，而非印迹调控区3＇-UTR则无明显变化，提示lgf-2r基因印迹调控区被破坏，很可能是导致克隆牛发育异常的一个重要原因。

英文摘要：

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.The 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 these 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 Igfi2r DMR2 was variable in different tissues, suggesting that the mechanism of gene imprinting was differently regulated in these tissues. The 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.