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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    • "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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