Methylation Status Of Imprinted Genes In Transgenic Somatic Cell Nuclear Transfer Cloned Sheep

At present, many transgenic cloned animals showed heterogeneous DNA methylation profiles. However, fewer reveals were well demonstrated both in normal development and in somatic cell nuclear transfer (SCNT) transgenic cloned sheep owe to limited resources of the transgenic cloned offspring, the ovine genome.In this study, in order to show the reprogramming degree of transgenic cloned lambs, we investigated DNA methylation patterns in CpG islands or differentially methylated regions of putative imprinted gene H19, IGF2, IGF2R, DLK1and Gtl2in transgenic cloned lambs. Different organs (hearts, lungs and muscles) from transgenic cloned lambs died shortly after birth and normal controls were researched. Firstly, paraffin and HE staining were used to study the morphological changes of transgenic cloned stillborn sheep; secondly, the five imprinted genes were used soft wares and on-line tools to analysis bio informatics, including gene structure, CpG Island, protein structure, phylogenetic tree; thirdly, we detect the DNA methylation status in CpG islands or DMR of five imprinted genes (H19, IGF2, IGF2R, DLK1, GTL2) in heart, lung, muscle, tissues of sheep; finally, We made a deep analysis of the DNA methylation data in test. The results were as follows:1. Staining slices of heart, lung and muscle of stillborn sheep were observed under light microscope and showed no significant abnormality, so the overall organ morphology was normal.2. Eleven CpG islands, including the upstream of gene, the exon, the intron and alternative splicing sites area, of five imprinted gene were as candidates for this research, in which we analyzed the methylation status of imprinted gene.3. The DNA methylation statuses of three CpG islands in H19were not significant differences between transgenic cloned lambs and controls, but there were special methylation statuses of several CpG sites in H19.4. The DNA methylation statuses of CpG sites in IGF2of transgenic cloned lamb were nearly same as controls; however, we still observed that the DNA methylation statuses of CpG sites in transgenic cloned sheep showed little higher in controls. And we found there were significant differences methylation among heart, lung and muscle in IGF2_R1of transgenic cloned sheep while controls were not.5. The DNA methylation statuses of IGF2R were significant differences among heart, lung and muscle in both test group and no differences between transgenic cloned and control sheep.6. There were no significant differences in DLK1_R1between transgenic cloned lambs and controls, and they are characterized by hypomethylation. But we observed that the DNA methylation of46CpG sites in DLK1_R2between transgenic cloned sheep and controls showed significant difference.7. In spite of, the DNA methylation statuses of GTL2were no significant differences in both test groups; we observed clear signs that most of CpG sties presented hypermethylation.In short, we received a bit of data to show that there were DNA methylation differences in imprinted genes between transgenic cloned and controls, but we still thought that there were not enough evidence to prove abnormal programming in transgenic clone sheep. We have still yet to find any evidence, such as increasing number of imprinted genes, adding more research material (including animal, and organization), gaining mRNA expression profiles of imprinted gene, detecting the DNA methylation level of different development period of sheep.