Production of cloned pigs by nuclear transfer of preadipocytes following cell cycle synchronization by differentiation induction.

Four methods of cell cycle synchronization of porcine preadipocytes for use as nuclear donors in somatic cell cloning were compared: serum starvation, differentiation induction, contact inhibition and roscovitine treatment. After three days of differentiation induction, the percentage of nuclear donor cells synchronized at the G0/G1 phase reached a peak value of 91.8%, which was significantly higher (P<0.05) than the percentage attained by serum starvation (84.9-89.8%), contact inhibition (78.3-83.7%) or roscovitine treatment (67.8-80.3%). Cell cycle synchronization by serum starvation, contact inhibition and roscovitine treatment all increased the percentage of apoptotic cells, while no increase was observed when the donor-cell cycle was synchronized by differentiation induction (Annexin V-positive: 15.7% to 19.3% vs. 7.7%, P<0.05; TUNEL-positive: 12.8% to 14.0% vs. 8.3%, P<0.05). Additionally, comparison of the in vitro development of nuclear transfer (NT) embryos formed from the nuclei of differentiation-induced or serum-starved preadipocytes revealed that, in both cases, a high proportion of embryos developed to the blastocyst stage (39.0 and 33.7%, respectively). In this study, NT embryos reconstructed with preadipocytes synchronized by differentiation induction were transferred to four recipient pigs, three of which gave birth to a total of 17 piglets (4.2%, 17/403). These results demonstrate that donor-cell cycle synchronization by differentiation induction enables effective production of cloned pigs. The findings also indicate that differentiation induction of multipotent cells is an excellent method of cell cycle synchronization that permits highly efficient synchronization of cells at the G0/G1 phase.