Document Detail


Synchronization of porcine fetal fibroblast cells with topoisomerase-inhibitor hoechst 33342.
MedLine Citation:
PMID:  11343846     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
B Kühholzer; R S Prather
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Animal reproduction science     Volume:  66     ISSN:  0378-4320     ISO Abbreviation:  Anim. Reprod. Sci.     Publication Date:  2001 Apr 
Date Detail:
Created Date:  2001-05-09     Completed Date:  2001-06-28     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  7807205     Medline TA:  Anim Reprod Sci     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  109-16     Citation Subset:  IM    
Affiliation:
Department of Animal Science, 162 Animal Science Research Center, University of Missouri-Columbia, 65211, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Benzimidazoles / pharmacology*
Cell Cycle / drug effects*
DNA Topoisomerases, Type I / antagonists & inhibitors*
Enzyme Inhibitors / pharmacology*
Fetus / cytology*
Fibroblasts / cytology*
G2 Phase
Gestational Age
Mitosis
Nuclear Transfer Techniques
Swine / embryology*
Grant Support
ID/Acronym/Agency:
RR 13438/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
0/Benzimidazoles; 0/Enzyme Inhibitors; 23491-52-3/HOE 33342; EC 5.99.1.2/DNA Topoisomerases, Type I

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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