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Cloning using somatic cell nuclear transfer (SCNT) has many potential applications such as in transgenic and genomic-edited animal production. Abnormal epigenetic reprogramming of somatic cell nuclei is probably the major cause of the low efficiency associated with SCNT. Strategies to alter DNA reprogramming in donor cell nuclei may help improve the cloning efficiency. In the present study, we aimed to characterize the effects of procaine and S-adenosyl-l-homocysteine (SAH) as demethylating agents during the cell culture of bovine skin fibroblasts. We characterized the effects of procaine and SAH on the expression of genes related to the epigenetic machinery, including the DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 alpha (DNMT3A), DNA methyltransferase 3 beta (DNMT3B), TET1, TET2, TET3, and OCT4 genes, and on DNA methylation levels of bovine skin fibroblasts. We found that DNA methylation levels of satellite I were reduced by SAH ( p  = 0.0495) and by the combination of SAH and procaine ( p  = 0.0479) compared with that in the control group. Global DNA methylation levels were lower in cells that were cultivated with both compounds than in control cells (procaine [ p  = 0.0116], SAH [ p  = 0.0408], and both [ p  = 0.0163]). Regarding gene expression, there was a decrease in the DNMT1 transcript levels in cells cultivated with SAH ( p  = 0.0151) and SAH/procaine (0.0001); a decrease in the DNMT3A transcript levels in cells cultivated with SAH/procaine ( p  = 0.016); and finally, a decrease in the DNMT3B transcript levels in cells cultivated with procaine ( p  = 0.0007), SAH ( p  = 0.0060), and SAH/procaine ( p  = 0.0021) was found. Higher levels of TET3 transcripts in cells cultivated with procaine ( p  = 0.0291), SAH ( p  = 0.0373), and procaine/SAH ( p  = 0.0013) compared with the control were also found. Regarding the OCT4 gene, no differences were found. Our results showed that the use of procaine and SAH during bovine cell culture was able to alter the epigenetic profile of the cells. This approach may be a useful alternative strategy to improve the efficiency of reprogramming the somatic nuclei after fusion, which in turn will improve the SCNT efficiency.

dna and cell biology

Procaine and S-Adenosyl-<scp>l</scp>-Homocysteine Affect the Expression of Genes Related to the Epigenetic Machinery and Change the DNA Methylation Status of<i>In Vitro</i>Cultured Bovine Skin Fibroblasts

Procaine and S-Adenosyl-l-Homocysteine Affect the Expression of Genes Related to the Epigenetic Machinery and Change the DNA Methylation Status ofIn VitroCultured Bovine Skin Fibroblasts