title: 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.
abstract: For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency.
authors.affiliation: National Agricultural Research Center for Kyushu Okinawa Region, Kumamoto, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yamanaka
authors.forename: Miki
authors.initials: M
authors.lastname: Sakatani
authors.forename: Kaiyu
authors.initials: K
authors.lastname: Kubota
authors.forename: Ahmed Zaky
authors.initials: AZ
authors.lastname: Balboula
authors.forename: Ken
authors.initials: K
authors.lastname: Sawai
authors.forename: Masashi
authors.initials: M
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D002417:Cattle; D065150:Cellular Reprogramming; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D004276:DNA, Satellite / Q000235:genetics; D015536:Down-Regulation; D004624:Embryo Transfer / Q000379:methods*; D047108:Embryonic Development; D005260:Female; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D053652:Nuclear Transfer Techniques; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D004276:DNA, Satellite; D034741:RNA, Small Interfering; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases