Synchronization of porcine fetal fibroblast cells with topoisomerase-inhibitor hoechst 33342

Proper synchronization of donor nuclei has been shown to have a major influ ence on the developmental potential of nuclear transfer embryos. In the pre sent study, a protocol was established to synchronize porcine fetal fibrobl asts in the G2 stage of the cell cycle. Cell cycle analyses were performed by flow cytometry. Cells were pre-synchronized by serum deprivation or aphi dicoline-treatment; then incubated in medium containing 0.1 mug/ml Hoechst 33342 (H342). The fluorochrome H342 has been shown to be a topoisomerase-in hibitor that can inhibit progression through the cell cycle. There was no s ignificant difference in the percentage of fibroblasts in G2/M whether cell s were pre-synchronized in medium supplemented with 0.1% serum for 48 h or 0.5% serum for 6 days. Compared with controls, pre-synchronization in early S-phase before incubation in H342 increased the proportion of G2/M fibrobl asts; also an increase from 0 and 6 versus 12h culture in complete medium b efore incubation in H342 resulted in an increased percentage of cells in G2 /M at the end of the synchronization period (9.3 and 13.1% versus 33.7%: P < 0.001). Neither an increase in the concentration of H342 (0.1-1.0 mug/ml) nor a longer exposure time (12h versus 18 h versus 21 h) increased the pro portion of G2/M fibroblasts. The protocol established in this study arreste d porcine fibroblasts reversibly in the G2/M-stage and is therefore suitabl e to provide synchronized cells for nuclear transfer experiments. (C) 2001 Elsevier Science B.V. All rights reserved.