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Developmental Capacity of Ferret Embryos by Nuclear Transfer Using G0/G1-Phase Fetal Fibroblasts¹

Departments of Anatomy & Cell Biology³ and Internal Medicine⁴ the Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases,⁵ College of Medicine, University of Iowa, Iowa City, Iowa 52242 Unite de Biologie du Developpement et Biotechnologie,⁶ Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, France

With the ultimate goal of establishing experimental protocols necessary for cloning ferrets, the present study has established parameters for the reconstruction of ferret embryos by nuclear transfer (NT) using G0/G1-phase donor fetal fibroblasts. Cumulus-oocyte complexes were harvested from superovulated ferrets and cultured in maturation medium for 24 h. Matured oocytes were then enucleated and injected with the fibroblast nuclei derived from 14–16-h serum-starved cells. Reconstructed embryos were then activated by a combination of electric pulses and chemical stimulations. Subsequently, the reconstructed and activated embryos were either cultured in vitro or transferred to pseudopregnant ferrets to evaluate their developmental capacity in vitro and in vivo. Our results demonstrated that 56.3% of reconstructed embryos (n = 187) cleaved, while 26.0% and 17.6% developed to morula and blastocyst phases in vitro, respectively. The blastocysts derived from NT embryos demonstrated normal morphology by differentially staining as compared to normal blastocysts developed in vivo following fertilization. In vivo developmental studies at 21 days posttransplantation demonstrated 8.8% of reconstructed embryos (n = 91) implanted into the uterine lining of recipients, while 3.3% formed fetuses. However, reconstructed embryos (n = 387) failed to develop to term (42 days). These results demonstrate donor nuclei of G0/G1-phase fetal fibroblast cells can be reprogrammed to support the development of reconstructed ferret embryos in vitro and in vivo; however, a significant third-trimester block occurs preventing full-term development.

¹ This research was funded by the Cystic Fibrosis Foundation, HL61234 (M.J.W.) and DK47967 (J.F.E.).

² Correspondence: John F. Engelhardt, Room 1-111 BSB, Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, 51 Newton Rd., Iowa City, IA 52242. FAX: 319 335 6581; john-engelhardt{at}uiowa.edu

This article has been cited by other articles:

				X J Yin, H S Lee, Y H Lee, Y I Seo, S J Jeon, E G Choi, S J Cho, S G Cho, W Min, S K Kang, et al. Cats cloned from fetal and adult somatic cells by nuclear transfer Reproduction, February 1, 2005; 129(2): 245 - 249. [Abstract] [Full Text] [PDF]