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a Department of Biotechnology, Institut für Tierzucht und Tierverhalten, Mariensee, D-31535 Neustadt, Germany
b Institute of Animal Physiology and Genetics, CZ-27721 Libechov, Czech Republic
c Department of Cell Biology, Fraunhofer Institut für Aerosolforschung und Toxikologie, D-30525 Hannover, Germany
The success of somatic nuclear transfer critically depends on the cell cycle stage of the donor nucleus and the recipient cytoplast. In this study we tested serum deprivation as well as two reversible cell cycle inhibitors, aphidicolin and butyrolactone I, for their ability to synchronize porcine fetal fibroblasts at either G0 stage or G1/S or G2/M transition. The synchronization efficiency of the various protocols was determined by fluorescence-activated cell sorting (FACS), cell proliferation assays, and semiquantitative multiplex reverse transcription-polymerase chain reaction detection of the cell cycle-regulated porcine Polo-like kinase mRNA (Plk-p). FACS measurements revealed that 66.6–73.3% of the porcine fetal fibroblasts were in G0/G1 stage (2C DNA content) in serum-supplemented medium. Short periods of 24–72 h of serum deprivation significantly increased the proportion of cells at G0/G1 phase to 77.9–80.2%, and mitotic activity had already terminated after 48 h. Prolonged culture in serum-deprived medium induced massive DNA fragmentation. Aphidicolin treatment led to an accumulation of 81.9 ± 4.9% of cells at the G1/S transition. Butyrolactone I arrested 81.0 ± 5.8% of the cells at the end of G1 stage and 37.0 ± 6.8% at the G2/M transition. The effects of both chemical inhibitors were fully reversible, and their removal led to a rapid progression in the cell cycle. The measurement of Plk-p expression allowed discrimination between the presumptive G0 phase induced by serum deprivation and the G1/S transition arrest achieved by chemical inhibitors. These data indicate that porcine fetal fibroblasts can be effectively synchronized at various cell cycle stages without compromising their proliferation capacity.
1 This research was supported by a grant from the German Federal Ministry for Education and Research (BMBF) and by the grant agency of the Czech Republic. Preliminary data were presented at the XXV Annual Meeting of the International Embryo Transfer Society, Quebec, Canada, January 1999.
2 Correspondence: Heiner Niemann; FAX: 5034 871 101; niemann{at}tzv.fal.de
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