Journal article
Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine
Journal of experimental zoology. Part A, Comparative experimental biology, Vol.303(12), pp.1126-1134
12/01/2005
DOI: 10.1002/jez.a.234
PMID: 16254916
Abstract
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.
Details
- Title: Subtitle
- Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine
- Creators
- Ziyi Li - Department of Anatomy & Cell Biology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA. ziyi-li@uiowa.eduXin ChenXingshen SunQi ZhouJuan ChenGregory H LenoJohn F Engelhardt
- Resource Type
- Journal article
- Publication Details
- Journal of experimental zoology. Part A, Comparative experimental biology, Vol.303(12), pp.1126-1134
- Publisher
- United States
- DOI
- 10.1002/jez.a.234
- PMID
- 16254916
- ISSN
- 1548-8969
- eISSN
- 1552-499X
- Grant note
- R01 DK047967 / NIDDK NIH HHS HL61234 / NHLBI NIH HHS DK47967 / NIDDK NIH HHS
- Language
- English
- Date published
- 12/01/2005
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Anatomy and Cell Biology; Radiation Oncology; Internal Medicine
- Record Identifier
- 9984025475802771
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