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Biology of Reproduction 64, 849-856 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Generation of Dwarf Goat (Capra hircus) Clones Following Nuclear Transfer with Transfected and Nontransfected Fetal Fibroblasts and In Vitro-Matured Oocytes1

C.L. Keefer2,a, H. Baldassarrea, R. Keystona, B. Wanga, B. Bhatiaa, A.S. Bilodeaua, J.F. Zhoua, M. Leduca, B.R. Downeyb, A. Lazarisa, and C.N. Karatzasa

a Nexia Biotechnologies Inc., b Ste Anne de Bellevue, Quebec, Canada H9X 3R2 Department of Animal Science, McGill University, Ste Anne de Bellevue, Quebec, Canada H9X 3V9

ABSTRACT

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4–8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

FOOTNOTES

First decision: 15 September 2000.

1 Preliminary reports of this work were presented at the 32nd Annual Meeting of the Society for the Study of Reproduction, July 1999, Pullman, WA, and the Transgenic Animal Research Conference, August 1999, Tahoe City, CA.

2 Correspondence: Carol L. Keefer, Nexia Biotechnologies Inc., 21025 Trans Canada Highway, Ste-Anne-de-Bellevue, PQ, Canada H9X 3R2. FAX: 514 457 6151; ckeefer{at}nexiabiotech.com




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