In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Meade, H.M. et al. in Gene expression systems: using nature for the art of expression, (eds Fernandez, J.M. & Hoeffler, J.P.) 399–427 (Academic Press, San Diego; 1998).
Clark, A.J. The mammary gland as a bioreactor: expression, processing, and production of recombinant proteins. J. Mammary Gland Biol. Neoplasia 3, 337–350 (1998).
Edmunds, T. et al. Transgenically produced human antithrombin—structural and functional comparison to human plasma-derived antithrombin. Blood 91, 4561–4571 (1998).
Wineland, N.E., Detwiler, L.A. & Salman, M.D. Epidemiologic analysis of reported scrapie in sheep in the United States: 1,117 cases (1947–1992). J. Am. Vet. Med. Assoc. 212, 713–718 (1998).
Ebert, K.M. & Schindler, J.E.S. Transgenic farm animals: Progress report. Theriogenology 39, 121– 135 (1993).
Wilkie, T.M., Brinster, R.L. & Palmiter, R.D. Germline and somatic mosaicism in transgenic mice. Dev. Biol. 118, 9–18 (1986).
Burdon, T.G. & Wall, R.J. Fate of microinjected genes in preimplantation mouse embryos. Mol. Reprod. Dev. 33, 436 –442 (1992).
Whitelaw, C.B.A., Springbett, A.J., Webster, J. & Clark, A.J. The majority of G0 transgenic mice are derived from mosaic embryos. Transgenic Res. 2, 29– 32 (1992).
Yong, Z. & Yuqiang, L. Nuclear-cytoplasmic interaction and development of goat embryos reconstructed by nuclear transplantation: production of goats by serially cloning embryos. Biol. Reprod. 58, 266–269 (1998).
Campbell, K.H.S., McWhir, J., Ritchie, W.A. & Wilmut, I. Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64–66, (1996).
Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H.S. Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813 (1997).
Wells, D.N., Misica, P.M., Day, T.A. & Tervit, H.R. Production of cloned lambs from an established embryonic cell line: a comparison between in vivo- and in vitro-matured cytoplasts. Biol. Reprod 57, 385–393 (1997).
Schnieke, A.E. et al. Human Factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278, 2130–2133 (1997).
Cibelli, J.B. et al. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280, 1256–1258 (1998).
Vignon, X. et al. Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells. C.R. Acad. Sci. 321, 735–745 (1998).
Wakayama, T., Perry, A.C.F., Zuccotti, M., Johnson, K.R., & Yanagimachi, R. Full-term development of mice from enucleated oocytes injected with cumulus cells nuclei. Nature 394, 369–374 (1998).
Kato, Y. et al. Eight calves cloned form somatic cells of a single adult. Science 282, 2095–2098 (1998).
Amills, M., Francino, O. & Sànchez, A. Nested PCR allows the characterization of TaqI and PstI RFLPs in the second exon of the caprine MHC class II DRB gene. Vet. Immunol. Immunopathol. 48, 313–321 (1996).
Bordignon, V. & Smith, L.C. Telophase enucleation: an improved method to prepare recipient cytoplasts for use in bovine nuclear transfer. Mol. Reprod. Dev. 49, 29– 36 (1998).
Laird, P.W. et al. Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19, 4293 (1991).
Aasen, E. & Medrano, J.F. Amplification of the ZFY and ZFX genes for sex identification in humans, cattle, sheep and goats. Bio/Technology 8, 1279– 1281 (1990).
Gavin, W.G. in Transgenic animals—generation and use (ed. Houdebine, L.M.) 19–21 (Harwood Academic Publishers, Amsterdam, 1996).
Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular cloning, a laboratory manual. (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1982).
Church, G.M. & Gilbert, W. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991– 1995 (1984).
Amills, M., Francino, O. & Sànchez, A. A PCR-RFLP typing method for the caprine Mhc class II DRB gene. Vet. Immunol. Immunopathol. 55, 255–260 (1996).
Ponce de Leon, F.A., Li, Y. & Weng, Z. Early and late replicative chromosomal banding patterns of Gallus domesticus. J. Hered. 83, 36– 42 (1992).
van de Corput, M.P.C., Dirks, R.W., van Gijlswijk, R.P.M., van de Rijke, F.M. & Raap, A.K. Fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection. Histochem Cell Biol. 110, 431– 437 (1998).
Klinger, K. et al. Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH). Am. J. Hum. Genet. 51, 55–65 (1992).
Di Berardino, D. et al. R-banding pattern of the prometaphase chromosomes of the goat. J. Hered. 78, 225–230 (1987).
Ryot, K.D., Vadnere S.V. & Prakash P. Hormonal induction of lactation and histomorphology of mammary glands in prepubertal goats. Indian J. Anim. Res. 10, 49–51 (1989).
This work was supported, in part, by a grant from the NIH (Small Business Innovation Research Program, R43 HD35395-01) to Genzyme Transgenics Corporation, and by Genzyme Transgenics Corporation. The authors gratefully acknowledge the help of M. Schofield, D.V.M., A. O'Coin, S. Bombard, N. Hawkins, S. Blash, R. Burns, and B. Kuehholzer. We wish to thank Gary Anderson for critical reading of the manuscript and helpful comments.
About this article
Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos
Journal of Dairy Science (2019)
Cellular and Molecular Life Sciences (2019)
Production of Transgenic Porcine Embryos Reconstructed with Induced Pluripotent Stem-Like Cells Derived from Porcine Endogenous Factors Using piggyBac System
Cellular Reprogramming (2019)
The Role of 5-aza-2′-Deoxycytidine on Methylation Status of Xist Gene in Different Genders of Buffalo (Bubalus bubalis) Bone Marrow Mesenchymal Stem Cells
Cellular Reprogramming (2019)
Improved preimplantation development of porcine somatic cell nuclear transfer embryos by caffeine treatment
Journal of Veterinary Science (2019)