Perfil transcricional de genes relacionados com a reprogramação da metilação do DNA em placenta de bovinos clones

Abstract

Cloning by somatic cell nuclear transfer (SCNT) is an assisted reproductive technique with several applications in livestock. However, it is still a technique that shows low efficiency due to the high frequency of fetal and placental abnormalities. The somatic genome must be correctly reprogrammed after nuclear transfer to achieve normal embryo development. However, in cloned embryos this reprogramming does not occur efficiently, leading to changes in DNA methylation patterns and consequently alterations in gene expression profile of important genes for embryo and foetal development. In this study, we determined the mRNA levels for six target genes (DNMT1, DNMT3A, DNMT3B, TET1, TET2 and TET3) related to DNA methylation reprogramming in the placental cotyledon of 13 calves produced by SCNT. Total RNA was extracted from the placental cotyledon samples and subjected to reverse transcription for real-time PCR analysis. Results showed different gene expression profiles, comparing placenta from cloned calves and placenta from animals produced by artificial insemination, except for TET3. The mRNA level of DNMT1 (p=0.0044), DNMT3A (p=0.0044) and TET2 (p=0.0044) were higher in the controls, while DNMT3B (p=0.0308) and TET1 (p=0.0308) were higher in the clones. In addition, it was identified that clones that died in the first week of life had differences in gene expression compared to the controls for the genes DNMT1 (p=0.0244), DNMT3A (p=0.0279), DNMT3B (p=0.0364), TET1 (p=0.0103) and TET2 (p=0.0184), while those that survived were different from controls only for TET1 (p=0.0344). These results showed that surviving clones are not only physically healthier but also relatively closer to the controls at the molecular level. This suggests that an adequate expression profile of enzymes related to epigenetic reprogramming is essential for the survival of cloned animals.