Somatic cell nuclear transferred (SCNT) animals are genetically identical to the donors; however, because of epigenetic abnormalities caused by incomplete reprogramming during nuclear transfer, the efficiency of SCNT is still very low. Monozygotic twins are also genetically identical, but it is reported that their epigenetic patterns on the genome, the so-called epigenome, are different. The epigenome is easily influenced by aging, environmental changes and nutrients, therefore these effects can be predicted by comparing epigenetic differences between genetically identical animals. Here we analysed DNA methylation levels of imprinted genes, which express in a parent-of-origin specific manner, in various tissues of cloned cows derived from the same donor cells. Imprinted gene expression is controlled by DNA methylation and other epigenetic modifications and abnormal expression/methylation patterns of imprinted genes have been observed in cloned animals. These alterations also occur during in vitro development of preimplantation embryos, which suggests that imprinted genes are easily influenced by environmental changes. Therefore, we chose H19 and PEG3 imprinted genes for the analysis to determine the epigenetic differences between individual cloned cows derived from the same donor cells. From 5 cloned and 5 non-cloned cows, we isolated DNA from 8 tissues (heart, lung, liver, kidney, spleen, intestine, muscle, and spinal cord) and analysed DNA methylation levels by bisulfite sequencing method. Briefly, genomic DNA was isolated by QIAGEN DNeasy Blood & Tissue Kit and bisulfite converted by QIAGEN EpiTect Bisulfite Kits (Qiagen, Valencia, CA). After amplification, the PCR products were cloned into TA vector and at least 10 clones were sequenced in each gene/sample. In every tissue analysed, the methylation levels largely differ among tissues and individuals. On average, the paternally imprinted gene H19 was 9.4 to 47.9% methylated (average 27.6 ± 10.3%) in clones and 0.5 to 69.8% methylated (average 29.0 ± 16.8%) in non-clones. The maternally imprinted gene PEG3 was 18.8 to 82.2% methylated (average 43.5 ± 15.8%) in clones and 8.0 to 98.7% (average 48.2 ± 18.8%) in non-clones. Even though there were large variations in DNA methylation levels, the variability tends to be low in clones compared to non-clones. More specifically, the variabilities of H19 methylation levels in spleen and intestine were significantly lower in clones than those in non-clones (32.3 ± 5.4% v. 27.0 ± 19.0% and 25.1 ± 4.2% v. 45.1 ± 14.3%, respectively, F-test; P < 0.05). These results suggest for the first time that epigenetic patterns in some tissues of both clones and non-clones are influenced by genetic background; however, mostly they are varied depending on non-genetic factors.
Abnormal DNA Methylation of Imprinted Loci in Human Preimplantation Embryos