Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine

Abstract

The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M‐phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28‐day fetuses and cultured for 1–30 days prior to NT. Fibroblast cultures were treated with 0.05 μg/ml of demecolcine for 3 hr or overnight (14–16 hr) after various times in culture to determine the optimal conditions for M‐phase synchronization. The percentage of G2/M‐phase cells in demecolcine‐treated cultures was significantly greater than that found in untreated cultures (PJ. Exp. Zool. 303A:1126–1234, 2005.