Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine

Department of Anatomy & Cell Biology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Journal of Experimental Zoology Part A Comparative Experimental Biology 12/2005; 303(12):1126-34. DOI: 10.1002/jez.a.234
Source: PubMed


The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M-phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28-day fetuses and cultured for 1-30 days prior to NT. Fibroblast cultures were treated with 0.05 microg/ml of demecolcine for 3 hr or overnight (14-16 hr) after various times in culture to determine the optimal conditions for M-phase synchronization. The percentage of G2/M-phase cells in demecolcine-treated cultures was significantly greater than that found in untreated cultures (P<0.05). Optimally synchronized M-phase fibroblasts were collected by mitotic shake-off and evaluated for their effectiveness in NT. M-phase somatic cell-derived NT embryos reconstituted by electrofusion or microinjection underwent implantation and formed fetuses at similar rates (5.4% vs. 3.4%, and 1.8% vs. 1.2%, respectively); however, no NT embryos developed to term. In summary, these data demonstrate two important points. First, demecolcine treatment effectively synchronizes ferret fibroblasts in M-phase of the cell cycle; and second, these somatic cells are capable of driving embryo development following NT. Our results should facilitate the development of cloned ferrets as an animal model for human lung disease such as influenza and cystic fibrosis.

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Available from: Ziyi Li, Aug 18, 2014
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    • "Researchers have used different approaches to synchronize the cell cycle of the donor cells, among them, cell confluency-contact inhibition (Hinrichs et al., 2006) and serum starvation (Li et al., 2003). In addition, chemical inhibitors have been used such as roscovitine (Gibbons et al., 2002), dimethyl sulfoxide (DMSO) (Hashem et al., 2007), butyrolactone I (Kues et al., 2000), aphidicolin (Collas et al., 1992), demecolcine (Li et al., 2005), Hoechst 33342 (Kühholzer and Prather, 2001), mimosine (Vacková et al., 2003) or colchicine (Lai et al., 2001) that result in cell cycle arrest at specific points. However, no work has been done on the control of the cell cycle stages in brown bear. "
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    ABSTRACT: The aim of this study was to assess by flow cytometry the cell cycle of brown bear fibroblast cells cultured under different growth conditions. Skin biopsies were taken in Cantabria (Spain) from a live, anaesthetized brown bear. DNA analysis was performed by flow cytometry following cell DNA staining with propidium iodide. Serum starvation increased (P<0.01) the percentage of G0/G1 phase cells (92.7+/-0.86) as compared to cycling cells (39.7+/-0.86) or cells cultured to confluency (87.3+/-0.86). DMSO included for 48h in the culture significantly increased (P<0.01) the percentage of G0/G1 phase of the cell cycle at all concentrations used and decreased percentages of S phase in a dose-dependent fashion. Roscovitine increased the G0/G1 phase of the cell cycle (P<0.01) at 15microM concentration. Interestingly, the G2/M stage significantly increased at 30 and 50microM compared to the control and 15microM (P<0.02). The cell cycle of brown bear adult fibroblast cells can be successfully synchronized under a variety of culture conditions.
    Cell Biology International 07/2008; 32(7):855-9. DOI:10.1016/j.cellbi.2008.02.005 · 1.93 Impact Factor
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    • "Ferret fetal fibroblasts were obtained from 28 dpc ( day post copulation ) fetuses derived from a Sable ( female ) × Cinnamon ( male ) mating ( Marshall Farms , North Rose , NY , USA ) , and cell lines were established as previously reported ( Li et al . , 2003 ) . Each fetus was treated individually . After karyotype analysis ( Li et al . , 2005a ) on individual cell lines , fibroblasts from 3 male fetuses were used for NT ( within 3 passages ) . Cumulus cells were collected from the COCs of sable coat - color ferrets , treated with 0 . 2% hyaluronidase / mPBS , and cultured in 10% FBS / DMEM medium for 3 – 7 days . The somatic cells were serum - starved for 24 h with DMEM conta"
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    ABSTRACT: Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.
    Developmental Biology 06/2006; 293(2):439-48. DOI:10.1016/j.ydbio.2006.02.016 · 3.55 Impact Factor
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