title: Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors.
abstract: 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.
authors.affiliation: Department of Biotechnology, Institut für Tierzucht und Tierverhalten, Mariensee, D-31535 Neustadt, Germany.
authors.forename: W A
authors.initials: WA
authors.lastname: Kues
authors.forename: M
authors.initials: M
authors.lastname: Anger
authors.forename: J W
authors.initials: JW
authors.lastname: Carnwath
authors.forename: D
authors.initials: D
authors.lastname: Paul
authors.forename: J
authors.initials: J
authors.lastname: Motlik
authors.forename: H
authors.initials: H
authors.lastname: Niemann
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D018797:Cell Cycle Proteins; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D053938:DNA Fragmentation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus / Q000166:cytology / Q000502:physiology*; D005347:Fibroblasts / Q000502:physiology; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014158:Transcription, Genetic / Q000235:genetics / Q000502:physiology; D000097869:Polo-Like Kinase 1
chemical_list: D015107:4-Butyrolactone; D016590:Aphidicolin; D001973:Bromodeoxyuridine; D018797:Cell Cycle Proteins; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D000097869:Polo-Like Kinase 1; C082868:butyrolactone I