title: Gene silencing of DNA methyltransferases by RNA interference in bovine fibroblast cells.
abstract: A highly methylated genome, like a somatic donor cell, is observed in somatic cell nuclear transfer (SCNT) embryos. The aberrant DNA methylation status causes global gene expression failure, resulting in low developmental competence of SCNT embryos. In addition, recent studies have uncovered the relationship between DNA methylation status and reprogramming efficiency. Because DNA methylation is performed by DNA methyltransferases (DNMTs), developing a technique which specifically inhibits DNMTs is necessary for further SCNT studies. In the present study, we examined the potential use of RNA interference for knockdown of DNMT mRNA in bovine fibroblast cells that were commonly used as karyoplast donors in SCNT studies. We designed three siRNAs corresponding to DNMT1, DNMT2 and DNMT3a mRNA. In Experiment 1, to optimize transfection conditions, fluorescence and cell viability after transfection were evaluated at different concentrations of transfection reagent using a FITC-labeled nonsilencing control siRNA. Although fluorescence was observed in all groups transfected except for the negative control group, transfection with a higher concentration of transfection reagent significantly decreased in cell viability (P<0.05). In Experiment 2, the amount of DNMT mRNA was measured by real-time PCR at 0, 48 and 96 h after siRNA transfection into the cells. The levels of each DNMT mRNA were significantly decreased at 48 and 96 h after transfection (P<0.01). Furthermore, decreased expression of DNMT1 protein was confirmed by western blotting. In Experiment 3, the DNA methylation statuses were analyzed in each of the siRNA-transfected groups. The DNMT1 siRNA-transfected group had a significantly decreased level of DNA methylation (P<0.05), but the other groups did not. Our data demonstrate that RNA interference with siRNA can be analyzed the function and expression of DNMT genes in bovine fibroblast cells. The present study provides useful information for further SCNT studies.
authors.affiliation: National Agricultural Research Center for Kyushu Okinawa Region, Egypt.
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Yamanaka
authors.forename: Ahmed Zaky
authors.initials: AZ
authors.lastname: Balboula
authors.forename: Miki
authors.initials: M
authors.lastname: Sakatani
authors.forename: Masashi
authors.initials: M
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D002417:Cattle; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation / Q000235:genetics*; D015254:DNA Modification Methylases / Q000235:genetics*; D005347:Fibroblasts / Q000201:enzymology; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D053652:Nuclear Transfer Techniques; D034622:RNA Interference
chemical_list: D004247:DNA; D015254:DNA Modification Methylases