Synchronization of Goat Fibroblast Cells at Quiescent Stage and Determination of Their Transition from G 0 to G 1 by Detection of Cyclin D 1 mRNA

Abstract

A number of studies have reported that donor cells consisting of serum starved cells, which are assumed to be at quiescence (G₀), or non-starved confluent cells or mitotic cells obtained by shake-off, both of which are assumed to be at G₁ phase, give better results in nuclear transfer (NT) than cells at other phases of the cell cycle. Whether G₀ or G₁ cells function better as donor cells is yet to be determined by detailed studies. The aims of this study were to analyze the cell cycle of goat transfected fibroblasts and determine the timing of transition from G₀ to G₁ by detecting G₁-specific marker, cyclin D₁ mRNA. Fluorescent-activated cell sorting (FACS) analyses of cells after 4 days of serum starvation showed that more that 90% of cells were in G₀/G₁. Additionally, detection of cyclin D₁ mRNA by northern blot analysis showed that 4-day serum starved quiescent cells started entering G₁ a few hours after addition of 10% serum to the medium. Taken together, the data indicated that serum starved transfected primary fibroblasts of adult goats experienced the G₀ to G₁ transition within 5 h of serum stimulation and were at the mid-G₁ stage within 10 h of serum stimulation.