B. Kuhholzer et Rs. Prather, Synchronization of porcine fetal fibroblast cells with topoisomerase-inhibitor hoechst 33342, ANIM REPROD, 66(1-2), 2001, pp. 109-116
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
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