Abstract
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.
Highlights
Two decades have passed since the first cloned mammal was born from an adult animal [1] and despite the somatic cell nuclear transfer (SCNT) technique having become a commercially available technique, its efficiency is still extremely low [2, 3].SCNT routinely involves the use of differentiated somatic cells, which display changed totipotency states through mechanisms dependent on epigenetic modifications [1, 4].Despite the successful cloning of several species, the use of differentiated somatic cells as donor nuclei is associated with a range of concerns, such as increased abortion rates, high embryonic lethality, and severe abnormalities in cloned foetuses and placentas in ruminants [5]
We characterised the effects of procaine and SAH on the expression of genes related to the epigenetic machinery, including the DNA methyltransferase 1 (DNMT1), DNMT3A, DNMT3B, TET1, TET2, TET3, and OCT4 genes, and on DNA methylation levels of bovine skin fibroblasts
We aimed to evaluate the effects of procaine and/or SAH during the in vitro culture of bovine skin fibroblasts on the global and specific DNA methylation patterns and on the mRNA levels of the genes encoding the epigenetic machinery, such as DNMT1, DNMT3A, DNMT3B, TET1, TET2, TET3, and the pluripotency gene OCT4
Summary
Two decades have passed since the first cloned mammal was born from an adult animal [1] and despite the somatic cell nuclear transfer (SCNT) technique having become a commercially available technique, its efficiency is still extremely low [2, 3].SCNT routinely involves the use of differentiated somatic cells, which display changed totipotency states through mechanisms dependent on epigenetic modifications [1, 4].Despite the successful cloning of several species, the use of differentiated somatic cells as donor nuclei is associated with a range of concerns, such as increased abortion rates, high embryonic lethality, and severe abnormalities in cloned foetuses and placentas in ruminants [5]. Some studies have reported that cloned embryos only partially demethylate their genomes and begin the de novo methylation process earlier than their counterparts [5, 7]. Epigenetic processes such as DNA methylation and demethylation are regulated by different groups of enzymes. Through early development, methylation is reduced on or removed from numerous sequences, and from the 8-16 cell stage, a new embryonic methylation pattern generated by the de novo methyltransferases DNA methyltransferase 3 alpha (Dnmt3a) and DNA methyltransferase 3 beta (Dnmt3b) is established [5, 7]. New patterns of DNA methylation on the genome are maintained by DNA methyltransferase 1 (DNMT1) and determine gene expression in embryo development [8]. The TET family contains the TET1, TET2, and TET3 dioxygenases, which enable the conversion of 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5-hmC)
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