Abstract
The current study was to investigate whether embryo or fetal fibroblast cells treated with 5-aza-2’-deoxyctidine (5-aza-dC) have a positive effect on the in vitro development of porcine parthenogenetic and cloned embryos. To this end, porcine fetal fibroblast cells were treated with different concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours), the results showed that DNA methylation in PRE-1 SINE region was gradually reduced over time in cells treated with 5-aza-dC. To determine the effect of 5-aza-dC on in vitro development of porcine activated oocytes, the parthenogenetic embryo was treated with 5-aza- dC. Notably, treatment with 5 nM 5-aza-dC for 1 hour led to a significant improvement in blastocyst development, compared with the control group. The effects of donor cell treatment with 5-aza-dC on porcine cloned embryos development were further examined by treating fetal fibroblast cells with various concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours). Exposure of cells in 5 nM 5-aza-dC for 1 - 20 hours led to a significant improvement in the percentage of developed blastocysts, while treatment with 500 nM 5-aza-dC did not affect blastocyst development, compared to untreated controls. These findings indicate that treatment of fetal fibro-blast cells with relatively low concentrations of 5-aza-dC for short exposure times improves subsequent blastocyst development of porcine cloned embryos.
Highlights
Somatic cell nuclear transfer (SCNT) has been successfully employed to generate cloned animals of various species [1]
Developmental failure of cloned embryos may be predominantly attributed to incomplete epigenetic reprogramming of donor genomic DNA [7,8], and low success rates of cloning and abnormal cloned offspring have been associated with abnormal epigenetic patterns in nuclear transfer (NT) embryos [5,8]
The methylation status of centromeric satellite and PRE-1 SINE sequences was determined in 5-aza-dCtreated fetal fibroblast cells (Figures 1 and 2)
Summary
Somatic cell nuclear transfer (SCNT) has been successfully employed to generate cloned animals of various species [1]. The efficiency of this technique was considered unsatisfactory due to poor in vitro embryonic development and the limited numbers of offspring obtained [4]. Epigenetic modifications, such as DNA methylation and histone acetylation, have been shown to regulate gene expression in higher organisms [5]. Developmental failure of cloned embryos may be predominantly attributed to incomplete epigenetic reprogramming of donor genomic DNA [7,8], and low success rates of cloning and abnormal cloned offspring have been associated with abnormal epigenetic patterns in nuclear transfer (NT) embryos [5,8]. The success rate of animal cloning may be improved by preventing or reversing epigenetic errors, such as DNA hypermethylation or histone hypoacetylation status [9,10]
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