Epigenetic status of buffalo fibroblasts treated with sodium butyrate a chromatin remodeling agent

摘要

The somatic cells having higher levels of DNA methylation and reducing it in donor cells before nuclear transfer (NT) by treating them with chemicals, may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Therefore, the present study was planned to understand mechanism of epigenetic changes in donor cells (buffalo fibroblasts) treated with different concentration of sodium butyrate (NaBu)-a chromatin remodeling agent. The cultured fibroblasts purity and lineage were confirmed by fibroblast specific protein and gene markers (Vimentin, Tubulin and Cytokeratin) at different passages using immuno-staining and qPCR respectively. The buffalo fibroblast cells were treated with 1, 3 and 5 mM of NaBu and observations were taken on their morphological changes, population doubling time and cell proliferation after 48 h of treatment. The epigenetic changes were observed using acetylation (H3K9ac) and methylation (H3K27me3) markers expression. The fibroblast cells derived from new born ear tissue started emerging and anchoring to cell culture flasks within 24 h and showed spindle-shaped morphology. The confluent culture was cryopreserved at different time interval. The post thaw culture behavior of the cryopreserved cells was also observed at different time interval and passages. The morphology of NaBu treated cells were changed with increase of dosages of NaBu treatment. The increase population doubling times and decreases the proliferation rate in the dose dependent manner. The NaBu treatment showed that the significantly increased the acetylation (H3K9ac) and methylation (H3K27me3) level over the control. Based on the observations of fibroblast characterization as well as epigenetic modifications of these cells after treatment with NaBu, results suggest that the cells may provide a useful approach for better epigenetic reprogramming in SCNT embryos.