PARENT SESSION
Preimplantation Embryo/Fetal Development
170
BOVINE CLONING USING ADULT DONOR CELLS TREATED WITH ROSCOVITINE.
Arat, Sezen1,3, Gibbons, John1, Rzucidlo, S. Jacek1, Miyoshi, Kazuchika1,4, Venable, Alison2, Waltenburg, Rachel1, Stice, Steven1, 1 3 4 2
ABSTRACT- The objective of this study was to examine the use of bovine donor cells treated with the cell cycle inhibitor, roscovitine, for cloning. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/ streptomycin, rIGF-1, bFSH, and bLH. Bovine granulosa cells were isolated from ovarian follicles and cultured in DMEM-F12 supplemented with 10% FBS at 37 oC in 5% CO2 in air. Prior to nuclear transfer (NT), a group of donor cells was cultured in media with 0.5 % serum for 4 days and another group of donor cells was exposed to 15 
M roscovitine for 24 hours. After NT and fusion, NT units were activated using a combination of calcium ionophore, cytochalasin B and cycloheximide and cultured for 7 days in BARC medium. Roscovitine treated, serum-starved and cycling control cells were analyzed to examine the phase of cell cycle by using flow cytometry. The roscovitine group had more cells in G1-G0 phase than the serum starved group and controls (82.4 ± 0.2% vs 76.7 ± 1.2% and 75 ± 0.2 %, respectively, p<0.05). There was not a significant difference between serum-starved cells and control cells in the G1/G0 phase. The percentage of cells in G2-M phase in the roscovitine group was significantly lower than serum-starved and controls (9.5 ± 0.1% vs 13.9 ± 0.5%, 16.5 ± 0.1%; respectively, p<0.05). Cleavage and blastocyst formation rates were similar between NT units derived from roscovitine (ros) treated or serum starved (ss) cells (cleavage: 54.3 ± 1.8% vs 57.7 ± 5.0%, blastocysts: 12.9 ± 0.5% vs 20.1 ± 4.7%; respectively). Following embryo transfer, there was not significant difference in fetal developmental rate to 30-70 days (ss: 23.6 ± 0.4%, ros: 27.8 ± 2.7%; respectively) or to 190 days (ss: 8.4 ± 5.9%, ros:12.5 ± 2.0%; respectively) between transferred blastocysts derived from serum starved or roscovitine treated cells, however; the development rate beyond 200 days in the roscovitine group (12.5 ± 2.0%) was significantly higher than the serum-starved group (3.4 ± 1.7%, p<0.05). These results indicated that roscovitine can arrest cells in the G1 phase of the cell cycle more effectively than serum-starvation and these cells can be used for production of cloned embryos and fetuses.
KEY WORDS: bovine, cloning, cell cycle, roscovitine