Caracterização epigenética do locus H19/IGF2 na placenta de bezerros clones

Abstract

Somatic Cell Nuclear Transfer (SCNT- Cloning) is a technique that is based on the production of genetically identical individuals. SCNT has potential application in several areas, such as transgenesis, preservation of endangered animals, animal production, basic sciences and human medicine. However, its efficiency is still low. Birth rates range from 0 to 10%. Studies suggest that the main cause of this low efficiency of the technique is an inadequate epigenetic reprogramming in the early stages of development. IGF2 and H19 genes are imprinted genes that are characterized by monoallelic expression and dependent on the allele's parental origin. These genes are related to embryonic development, placenta and fetal growth. The objective of this study was to characterize the methylation pattern of an exon 10 region of the IGF2 gene and the H19 gene Imprinting Control Region (ICR) in the placenta of cloned animals. For this study female and male cloned calves presenting different phenotypes were used. Genomic DNA from placenta of these animals was isolated, then treated with sodium bisulfite and amplified to the ICR/H19 and IGF2 loci. PCR products were cloned into competent bacteria and finally sequenced. A significant difference was found between controls and clones with healthy phenotypes for the ICR/H19 region. In this region controls showed a hemimethylated pattern, as predicted in the literature due to this region has an imprinted control, while clones were showed less methylated. For the IGF2, no significant differences were found between controls and clones, and between clones with aberrant and healthy phenotypes. These results suggest that different genomic regions in the genome may be independently reprogrammed and that failures in reprogramming the DNA methylation patterns of imprinted genes may be one of the causes of the low efficiency of SCNT.