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Cell cycle synchronisation of porcine primary fibroblasts: Effects of starvation and reversible cell cycle inhibitors
Theriogenology
205
CELL CYCLE SYNCHRONISATION OF PORCINE PRIMARY FIBROBLASTS: EFFECTS OF STARVATION AND REVERSIBLE CELL CYCLE INHIBITORS W. A. Kues ~'s, M. Anger2, J.W. Carnwath ~, J. Motlik2, H. Niemann I and D. Paul 3 qnstitut ftir Tierzucht and Tierverhalten, Mariensee, D-31535 Neustadt, Germany; 2Institute of Animal Physiology and Genetics, 27721 Libechov, Czech Republic; 3Fraanhofer Institut f'tir Aerosolforschung und Toxikologie, 30525 Hannover, Germany Recent advances in nuclear transfer techniques highlight the potential use of somatic cells to genetically engineer farm animals. To apply this technology for genetically engineered pigs, the use of porcine fetal fibroblasts as nuclei donors is currently being investigated. It has been suggested that cell cycle stage of the donor nuclei is critical for nuclear transfer and that either Go or G 1 stages are preferential for this purpose. Therefore, we measured the cell cycle stage of porcine fetal fibroblasts under different growth conditions by fluorescence-activated cell sorting (FACS) analysis and tested the utility of two reversible cell cycle inhibitors, aphidicolin and butyrolactone I, for cell cycle synchronisation. In parallel, expression of porcine POLO-like kinase (Plkl), a major regulator of cell cycle was determined by semi-quantitative RT-PCR. Primary fibroblasts were prepared from day 25 pig fetuses and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS), 1 mM L-glutamine and antibiotics. Approximately 5 x 106 vital cells were isolated per embryo. The cells of one preparation were pooled and expanded for at least two passages. Cells of passage 2 were then trypsinised and stored as frozen samples. Control experiments had shown that fibroblasts could be expanded up to twenty passages, with subpassaging intervals of 4 to 5 days. For each series of experiments, porcine fibroblasts of the same batch (passage 3) were grown to 80% confluency. In experiment I, unsynchronised primary fibroblasts were cultured in DMEM with 10% FCS, harvested after 1, 2 or 3 days and processed for FACS analysis, respectively. FACS measurements showed that 70-76% of the cells were in G 1 stage, semi-quantitative RT-PCR revealed moderate levels of Plkl. In experiment II, the fibroblasts were synchronised by serum reduction to 0,2% FCS for up to 3 days. FACS analysis detected an increase of cetls in Go]G1 stages to app. 83% after 3 days serum reduction compared to unsynchronised cells. The amount of Plkl-mRNA decreased dramatically after 1 day and became undetectable after two days of serum reduction. In experiment III, cells were 5rst starved (2 days, 0.2% FCS), the FCS concentration was then increased to 10% for 14 hours. This treatment induced cells to progress in a synchronised manner from Go/G ~ to S phase. Addition of 6 mM aphidicolin, a DNA replication inhibitor, completely arrested cells at the boundary of GI to S phase. Washing of the cells and culture without aphidicolin, again led to cell cycle progression. In experiment IV, cells synchronised by serum reduction for 2 clays were cultured in the presence of 100 mM butyrolactone I, an inhibitor of cyclin-dependent kinases. This treatment reversibly inhibited cell cycle progression from G~ to S in about 85% of primary fibroblasts. In summary, pig fetal fibroblasts were sucessfully synchronised at G0/G ~ (serum starvation) stages and at the boundary from G~ to S (aphidicolin or butyrolactone I). This was confn-rned by FACS analysis and by semi-quantitative RT-PCR of cell cycle-regulated Plkl. Thus this approach can be used to characterise primary cell lines and to increase specific cell cycle stages for the subsequent use in nuclear transfer.
205
CELL CYCLE SYNCHRONISATION OF PORCINE PRIMARY FIBROBLASTS: EFFECTS OF STARVATION AND REVERSIBLE CELL CYCLE INHIBITORS W. A. Kues ~'s, M. Anger2, J.W. Carnwath ~, J. Motlik2, H. Niemann I and D. Paul 3 qnstitut ftir Tierzucht and Tierverhalten, Mariensee, D-31535 Neustadt, Germany; 2Institute of Animal Physiology and Genetics, 27721 Libechov, Czech Republic; 3Fraanhofer Institut f'tir Aerosolforschung und Toxikologie, 30525 Hannover, Germany Recent advances in nuclear transfer techniques highlight the potential use of somatic cells to genetically engineer farm animals. To apply this technology for genetically engineered pigs, the use of porcine fetal fibroblasts as nuclei donors is currently being investigated. It has been suggested that cell cycle stage of the donor nuclei is critical for nuclear transfer and that either Go or G 1 stages are preferential for this purpose. Therefore, we measured the cell cycle stage of porcine fetal fibroblasts under different growth conditions by fluorescence-activated cell sorting (FACS) analysis and tested the utility of two reversible cell cycle inhibitors, aphidicolin and butyrolactone I, for cell cycle synchronisation. In parallel, expression of porcine POLO-like kinase (Plkl), a major regulator of cell cycle was determined by semi-quantitative RT-PCR. Primary fibroblasts were prepared from day 25 pig fetuses and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS), 1 mM L-glutamine and antibiotics. Approximately 5 x 106 vital cells were isolated per embryo. The cells of one preparation were pooled and expanded for at least two passages. Cells of passage 2 were then trypsinised and stored as frozen samples. Control experiments had shown that fibroblasts could be expanded up to twenty passages, with subpassaging intervals of 4 to 5 days. For each series of experiments, porcine fibroblasts of the same batch (passage 3) were grown to 80% confluency. In experiment I, unsynchronised primary fibroblasts were cultured in DMEM with 10% FCS, harvested after 1, 2 or 3 days and processed for FACS analysis, respectively. FACS measurements showed that 70-76% of the cells were in G 1 stage, semi-quantitative RT-PCR revealed moderate levels of Plkl. In experiment II, the fibroblasts were synchronised by serum reduction to 0,2% FCS for up to 3 days. FACS analysis detected an increase of cetls in Go]G1 stages to app. 83% after 3 days serum reduction compared to unsynchronised cells. The amount of Plkl-mRNA decreased dramatically after 1 day and became undetectable after two days of serum reduction. In experiment III, cells were 5rst starved (2 days, 0.2% FCS), the FCS concentration was then increased to 10% for 14 hours. This treatment induced cells to progress in a synchronised manner from Go/G ~ to S phase. Addition of 6 mM aphidicolin, a DNA replication inhibitor, completely arrested cells at the boundary of GI to S phase. Washing of the cells and culture without aphidicolin, again led to cell cycle progression. In experiment IV, cells synchronised by serum reduction for 2 clays were cultured in the presence of 100 mM butyrolactone I, an inhibitor of cyclin-dependent kinases. This treatment reversibly inhibited cell cycle progression from G~ to S in about 85% of primary fibroblasts. In summary, pig fetal fibroblasts were sucessfully synchronised at G0/G ~ (serum starvation) stages and at the boundary from G~ to S (aphidicolin or butyrolactone I). This was confn-rned by FACS analysis and by semi-quantitative RT-PCR of cell cycle-regulated Plkl. Thus this approach can be used to characterise primary cell lines and to increase specific cell cycle stages for the subsequent use in nuclear transfer.