88 CELL CYCLE SYNCHRONIZATION OF FIBROBLASTS DERIVED FROM TRANSGENIC CLONED CATTLE EAR SKIN: EFFECTS OF SERUM STARVATION, ROSCOVITINE, AND CONTACT INHIBITION

Abstract

Cell cycle stage plays a critical role in somatic cell nuclear transfer (SCNT), and G0/G1 stage cells are preferred nuclear donors in attempts to produce cloned livestocks. Enhancement of survivability of cloned calves by roscovitine (the cyclin-dependent kinase-2 inhibitor) has been shown (Gibbons et al. 2002 Biol. Reprod. 66, 895–900). The purpose of this study was to evaluate the effects of serum starvation, roscovitine, and contact inhibition on cell cycle synchronization at the G0/G1 stage of transgenic cloned bovine ear skin-derived fibroblasts. The cell line was established from a cloned calf expressing green fluorescent protein (GFP). Data were analyzed by using SAS (8.0) with ANOVA (SAS Institute, Inc., Cary, NC, USA). At passage 2-6, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) in T-25 culture flasks from immediately after subculture until monolayer cells reached 90% confluence at 39°C, under 5% CO₂ in humidified air. Then cells grown in different flasks were randomly distributed to groups: Serum starvation (SS, culture medium changed to DMEM + 0.5% FBS), roscovitine (R, cultured in DMEM + 10% FBS + 15 µM roscovitine), and contact inhibition (CI, DMEM + 10% FBS). From Day 1 to Day 5 after treatment, 3 flasks of cells from each group were subjected to fixation and staining every day, followed by determination of cell cycle stage with Becton Dickinson FACScan (Kues et al. 2000 Biol. Reprod. 62, 412–419). At least 3 replicates were performed for each determination. The results showed that in the cycling fibroblasts (50–60% confluence), 59.29% of the cells were at the G0/G1 phase. For the SS group, the proportion of G0/G1 cells was significantly higher for treatment lasting 3 days, 4 days, and 5 days than for treatment lasting 1 day or 2 days (91.5%^c, 91.7%^c, and 93.5%^c vs. 90.1%^b and 88.8%^a, respectively; P < 0.05). No statistical differences were observed among 3-day, 4-day, and 5-day treatments. For the R group, one-day treatment was significantly lower in synchronizing cells to the G0/G1 stage than that from Day 2 to Day 5 (86.51%^a vs. 91.1%^a, 90.1%^a, 89.4%^a, and 91.3%^b, respectively; P < 0.05) during which similar rates of G0/G1 phase were observed. For the CI group, an increase of G0/G1 stage cells was found after 3 days of CI (from 89.4%^a for Day 1 and 90.4%^ab for Day 2, to 91.4%^bc for Day 3; P < 0.05), which was similar to 4 days (91.6%^bc) and 5 days (92.1%^c) for the CI group. We also found that the efficiency of obtaining G0/G1 phase cells was lower when roscovitine was employed to synchronize the cell cycle than when the SS and CI methods were used (89.7%^b vs. 91.1%^a and 91.0%^a, respectively; P < 0.05), after analysis of the most effective treatment duration of each group. Our data indicate that in attempts to harvest adequate G0/G1-stage cells for re-cloning of transgenic cattle from fibroblasts established from ear skin of a newborn transgenic cloned calf, 3 days of treatment is enough regardless of the method (SS, R, or CI). Further research is needed to evaluate the developmental competence of embryos cloned from cells prepared by the abovementioned treatments. [^a-c mean statistical differences; P < 0.05.]