PSXVI-12 Long term effects of cloning by nuclear transfer on monocytes methylome in Dairy Cattle.

摘要

To produce embryos by nuclear transfer, it needs a great epigenetic reprogramming of somatic nucleus into embryonic nucleus: this reprogramming is highly dependent of oocyte capacity. Thus, each nucleus is placed in a different environment as soon as the oocyte-somatic cell fusion. This way, the cloned animals obtained using the same fibroblast line, have the same nuclear genotype, but are indubitably epigenetically different . DNA methylation, is one of the more important epigenetic modification analyzed contributes play a role in regulating gene expression and consequently in different biological processes, such as those involved in health and disease. In this study, we analyzed the genome-wide DNA methylation of a purified subpopulation of immune cells, such as monocytes from cloned and controls Holstein (n=5 per group). Born in an experimental INRA’s farm the cows were breed exactly under the same conditions limiting environmental changes. All growth, health and reproductive information has been collected. Cows were hormonally synchronized, the ovulation detected by ultrasonography and blood samples collected at D15 of cycle. Monocyte purification was performed from peripheral blood mononuclear cells obtained by Ficoll gradient, using CD14+ antibody beads. Genome-wide DNA methylation was analyzed by Reduced Representation Bisulfite Sequencing (RRBS; [2]). After sequencing, the reads were analyzed using our dedicated pipeline ( ). After mapping to the reference genome (UMD3.1 assembly), around of 40% of uniquely mapped reads were obtained. Only CpGs covered between 10X and 500X were analyzed and used to calculate a DNA methylation score which was not different between cloned and control groups. However, PCA described a clear separation between cloned and control groups. Differential analysis of the 10 libraries provided differentially methylated cytosine (DMCs) between the two groups, corresponding to DMRs. This study therefore highlights CpGs and regions displaying a specific methylation pattern in cloned cows.