56 EFFECTS OF KNOCKDOWN OF DNA METHYLTRANSFERASE 1 BY RNA INTERFERENCE ON IN VITRO DEVELOPMENT AND DNA METHYLATION STATE IN BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Abstract

Reprogramming of epigenetic modification is a necessary process during mammalian development, which is aberrant in somatic cell nuclear transfer (SCNT) embryos. Previous study has demonstrated that an abnormal state of genomic hypermethylation is consistently observed in SCNT embryos (Kang et al. 2001 Nat. Genet. 28, 173–177). On the other hand, small interfering RNAs (siRNAs) are identified molecules shown to silence genes via targeted mRNA degradation and are widely used in molecular and cellar research (Hannon GJ 2002 Nature 418, 244–251). Thus, knockdown of the expression of genes related to epigenetic modifications by siRNA may be used to alter epigenetic modifications in SCNT embryos. In the present study, we investigated the effects of knockdown of DNA methyltransferase 1 by siRNA on in vitro development, gene expression, and DNA methylation state of bovine SCNT embryos. In vitro matured oocytes were enucleated, fused with bovine fibroblasts and then activated, the resultant SCNT embryos were divided into three groups; control, non-treated group; sham-NT, H₂O injected group; and siRNA-NT, siRNA injected group. The siRNA corresponding to DNA methyltransferases 1, which is the enzyme responsible for maintaining DNA methylation patterns, was designed and injected into the cytoplasm of SCNT embryos. All embryos were cultured in CR1aa + 5% FCS and assessed the rates of cleavage and blastocyst formation on Days 2 and 8, respectively. All data were obtained from more than 5 replicates. Developmental percentage data were analyzed by chi-square tests (P < 0.05). Other data were analyzed with ANOVA followed by Fisher’s protected least significant difference (P < 0.05). The developmental rate to blastcysts in siRNA-NT group (38.7%; 111/287) was significantly higher (P < 0.05) than those of control (28.8%; 121/420) and sham groups (30.5%; 92/302). To estimate the effect of siRNA injection on gene expression, we sampled embryos at 48 h after culture and measured the amount of DNA methyltransferase 1 mRNA expression by real-time PCR. The amount of DNA methyltransferase 1 mRNA was significantly less (P < 0.05) than those of control and sham-NT groups. Finally, the levels of DNA methylation at satellite I region were analyzed by COBRA method in blastosyst stage embryos. The level of DNA methylation of blastocysts in siRNA-NT groups was significantly less (P < 0.05) than those of control and sham-NT and also similar to that of IVF blastocysts. In the present study, we showed that gene silencing of DNA methyltransferase 1 by siRNA enhanced the in vitro development of SCNT embryos and decreased the level of DNA methylation which was equivalent to IVF embryos. These findings suggest that knockdown of specific genes related epigenetic modifications by RNA interference may alter abnormal epigenetic reprogramming with the resultant improvement for subsequent development of SCNT embryos.