30 INHIBITION OF DNA METHYLTRANSFERASE 1 EXPRESSION IN BOVINE FIBROBLAST CELLS 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 (NT). DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. NT utilizing cell fusion introduces the somatic form of DNMT1 (DNMT1s), which is not normally present in preimplantation embryos and could perpetuate the somatic-like methylation patterns observed in early cloned embryos. The objective of this study was to decrease the level of DNMT1s in bovine fibroblasts using siRNA, prior to their use as donor cells. Fetal fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO₂ at 37°C. The transfection efficiency of different ratios of siRNA concentrations and transfection reagent (µg:µL; 1:3, 1:6, and 1:9) as well as siRNA concentrations (0.5, 1.0, and 1.5 µg) were determined using fluorescein isothiocyanate (FITC)-labeled control siRNA and fluorescence analyzed by flow cytometry. A DNMT1s-specific siRNA was used to transfect cells at 50 and 80% of confluence. A non-silencing siRNA was used as a negative control. The expression patterns of DNMT1s were characterized by Q-PCR using the δδC_T method. ANOVA was used to detect differences in transfection efficiency and gene expression. The combination of 1.0 or 1.5 µg siRNA and a 1:6 siRNA to transfection reagent ratio produced the highest transient transfection rates without affecting cell viability. Cells treated at 50% confluence with 1.5 µg of DNMT1s-specific siRNA at a 1:6 ratio showed 80% less DNMT1s mRNA than cells treated with non-silencing siRNA. At 8 h post-transfection (PT) these cells displayed vacuole-like structures within the cytoplasm, stopped dividing, and died approximately 24 h PT. Cells treated at 80% confluence with 1.0 µg DNMT1s-specific siRNA at a 1:6 ratio resulted in 57, 66, 24, 50, 22, 62, 64, and 56% less DNMT1s than control cells at 4, 6, 8, 10, 12, 24, 48, and 72 h PT, respectively, but without the cytotoxic effects observed when cells at 50% of confluence were treated under the same transfection conditions. These data indicate that optimization of cell density, siRNA concentration, and the ratio between siRNA concentration and transfection reagent are required to decrease the levels of DNMT1s without causing deleterious effects to the cells. However, levels of DNMT1s mRNA can be effectively reduced using siRNA; protein analysis is required to determine if reduction of the transcript results in lower levels of the protein. Subsequent use of these cells for NT will provide insight as to how the presence of this enzyme affects reprogramming in early cloned embryos.

This study was supported by Louisiana State University Board of Regents.