Cell Cycle Synchronization of Bison (Bos Gaurus) Fibroblasts Derived from Ear Piece Collected Post‐mortem

Abstract

Efficiency of the technique of somatic cell nuclear transfer critically depends on the cell cycle phase compatibility between the donor somatic cell nucleus and recipient cytoplasm. In this study, attempts were made to optimize conditions for cell cycle synchronization of bison ear fibroblasts at G0/G1 using different approaches such as using cells in confluency, after contact inhibition, serum starvation or treatment with dimethyl sulfoxide (DMSO) (0.5%, 1.0% and 2.0%), sodium butyrate (NaBu) (0.5, 1.0 and 2.0 mm), cytochalasin-B (CB) (7.5 μg/ml), cycloheximide (CHX) (7.5 μg/ml) and 6-dimethyl aminopurine (6-DMAP) (2.0 mm). A small piece of an ear of an adult female bison collected post-mortem 10 h after death was used for the preparation of fibroblast cells. The synchronization efficiency was determined by fluorescence-activated cell sorting. Higher proportion of G0/G1 phase was obtained when cells were subjected to serum starvation for 48 h (85.4%). Sodium butyrate had no effect on synchronization of cells at G0/G1 when the cells were treated for 24 and 48 h. Similarly, DMSO (0.5% and 1.0%) had also no effect on the proportion of cells at G0/G1 for 24 and 48 h. The synchronization ability of CB, CHX and 6-DMAP at G0/G1 phase was equally effective when cells were treated for 4 h (68.5%, 68.7% and 67.4%) and 24 h (67.8%, 66.1% and 67.5%). In conclusion, this study shows that cells subjected to serum starvation for 24-48 h or confluent monolayer, or cycling cells treated with 1.0% DMSO or 2.0 mm NaBu for 24 h showed best synchronization in G0/G1 phase of cell cycle.