Synchronization of porcine fetal fibroblast cells with topoisomerase-inhibitor hoechst 33342.
Proper synchronization of donor nuclei has been shown to have a major influence on the developmental potential of nuclear transfer embryos. In the present study, a protocol was established to synchronize porcine fetal fibroblasts in the G2 stage of the cell cycle. Cell cycle analyses were performed by flow cytometry. Cells were pre-synchronized by serum deprivation or aphidicoline-treatment; then incubated in medium containing 0.1 microg/ml Hoechst 33342 (H342). The fluorochrome H342 has been shown to be a topoisomerase-inhibitor that can inhibit progression through the cell cycle. There was no significant difference in the percentage of fibroblasts in G2/M whether cells were pre-synchronized in medium supplemented with 0.1% serum for 48h 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 fibroblasts; also an increase from 0 and 6 versus 12h culture in complete medium before 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 microg/ml) nor a longer exposure time (12h versus 18h versus 24h) increased the proportion of G2/M fibroblasts. The protocol established in this study arrested porcine fibroblasts reversibly in the G2/M-stage and is therefore suitable to provide synchronized cells for nuclear transfer experiments.
Kühholzer
B
B
Department of Animal Science, 162 Animal Science Research Center, University of Missouri-Columbia, 65211, USA.
Prather
R S
RS
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005333:Fetus / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology*; D016195:G2 Phase; D005865:Gestational Age; D008938:Mitosis; D053652:Nuclear Transfer Techniques; D013552:Swine / Q000196:embryology*; D059004:Topoisomerase I Inhibitors
D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; C017807:bisbenzimide ethoxide trihydrochloride