Inhibition of DNA methyltransferase 1 expression in bovine fibroblast cells used for nuclear transfer

Abstract

The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer. DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. The presence of DNMT1 transcript in the donor cell may contribute to perpetuation of the highly methylated status of the somatic nuclei in cloned embryos. The objective of the present study was to determine the methylation pattern of cloned embryos reconstructed with cells treated with DNMT1-specific small interfering RNA (siRNA). Bovine fibroblasts were transfected with a DNMT1-specific siRNA under optimised conditions. The expression patterns of DNMT1 were characterised by Q-PCR using the DeltaDeltaC(T) method. The level of DNMT1 was successfully decreased in bovine fibroblast cells using a DNMT1-specific siRNA. Additionally, reduction in the expression of DNMT1 mRNA and DNMT1 protein led to a moderate hypomethylation pattern in the siRNA-treated cells. The use of siRNA-treated cells as donor nuclei during nuclear transplantation induced a reduction in methylation levels compared with controls but did not reduce methylation levels to that of IVF embryos. Further studies are required to determine if this level of reduced methylation is sufficient to improve subsequent development.

Effects of Downregulating DNA Methyltransferase 1 Transcript by RNA Interference on DNA Methylation Status of the Satellite I Region and In Vitro Development of Bovine Somatic Cell Nuclear Transfer Embryos

“…Incubation with anti-DNMT1 antibody produced a band of approximately 180 kDa ( Fig. 2A) as previously described [33]. The amount of DNMT1 protein in siRNA-NT embryos at 24, 48 and 72 h after in vitro culture was significantly lower (P<0.05) than in the sham-NT embryos at the same time point (Fig.…”

“…Since knockout of the DNMT1 gene has been shown to be lethal during embryonic development in mice [34,35], we decided to use a temporal and reversible method for specific inhibition of the DNMT1 gene in this study. In addition, although previous studies have reported the knockdown of DNMT1 mRNA by introduction of siRNA in donor cells [33,36,37], the transfection efficiency is approximately 70-80%. Thus, it is difficult to select only the transfected donor cell during nuclear transfer in the case of siRNA transfection into donor cells during NT.…”

“…This discrepancy might be due to the transfection efficiency. As mentioned above, the efficiency of transfection into somatic cells was approximately 70-80% in previous studies [33,36,37]. Thus, the use of untransfected cells as donor cells may affect the experimental results.…”

“…Similarly, Wee et al [11] reported that treatment of donor cells with Trichostatin A, an inhibitor of histone deacetylase, prior to NT decreased levels of DNA methylation in the satellite I region and improved early developmental competence of bovine NT embryos. In contrast, Giraldo et al [33] reported that in vitro development of NT embryos does not increases when the donor cells are decreased by siRNA transfection. This discrepancy might be due to the transfection efficiency.…”

Probability, odds and random chance: the difficult task of modulating the epigenetic profile of cloned embryos

“…In fact, global genomic reprogramming occurs rapidly after fertilization, with the degree of DNA methylation decreasing in about 30% of the average level observed in somatic cells (Bird, 2002). This fact is interesting because when demethylation was experimentally induced below such values, a negative interference in embryonic development was observed, showing that a certain degree of methylation needs to be maintained in the genome (Giraldo et al, 2009). Following the reduction in DNA methylation during the first cleavages, a de novo methylation pattern is promoted by DNMT3a, 3b and 3L (Fig.…”