Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells

Normal development of nuclear transfer embryos is thought to be dependent o n transferral of nuclei in GO or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast ce lls cultured under a variety of cell cycle-arresting treatments. This was a chieved by using flour cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in CO, G 1, S, and G2+M phases of the cell cycle. Cultures that were serum starved f or 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0.7) a nd G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0 +G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0. 05) G0+G1 percentages (85.1 +/- 2.8) but did not increase GO percentages (6 .0 +/- 5.3) compared to those in cycling cultures. Separate assessment of s mall-, medium-, and large-sized cells showed that as the cell size decrease d from large to small, percentages of cells in G0+G1 and GO alone increased (p < 0.05). we found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starv ed cells in G0+G1 and GO alone, respectively. Cultures were also treated wi th cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchic ine (0.5 mu M) increased percentages of cells in CO (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transf er procedures.