Production of transgenic porcine blastocysts by nuclear transfer

• Published in: Molecular reproduction and development 2000-06, Vol.56 (2), p.145-148
• Main Authors: Kühholzer, B., Tao, T., Machaty, Z., Hawley, R.J., Greenstein, J.L., Day, B.N., Prather, R.S.
• Format: Article
• Language: English
• Subjects: Animals; Animals, Genetically Modified; Biological and medical sciences; Blastocyst - physiology; Embryology: invertebrates and vertebrates. Teratology; Female; fetal fibroblasts; Fibroblasts; Fundamental and applied biological sciences. Psychology; General aspects; General aspects. Development. Fetal membranes; in vitro; Kanamycin Kinase; Microinjections; neomycin; nuclear transfer; Oocytes - physiology; Parthenogenesis; pig; Receptor, Insulin - genetics; Swine; thimerosal
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/(SICI)1098-2795(200006)56:2<145::AID-MRD4>3.0.CO;2-8

In this study the in vitro development of porcine nuclear transfer (NT) embryos was investigated. Transgenic fetal fibroblast cells that were frozen after 5 days of serum starvation were injected immediately after thawing into enucleated metaphase II (MII) oocytes. Reconstructed embryos were activated by incubation in 200 μM thimerosal followed by a 30‐min treatment of 8 mM DTT. The embryos were subsequently cultured in NCSU23, supplemented with 4 mg/ml BSA for 7 days. The actual cleavage rate (embryos showing ≥2 nuclei) in 6 replicates was 33% (ranging from 15% to 50%). Three blastocysts with cell numbers of 14, 15, and 18 were obtained. The blastocyst rate was significantly lower for NT embryos as opposed to parthenogenetically activated embryos (1% vs 5%; P < 0.05). The neomycin‐resistance gene was amplified by PCR in all three NT embryos, indicating their origin from the injected transgenic fibroblasts. Efforts are now being directed in improvements in the nuclear transfer technology, whereby viable fetuses or offspring can be produced from these NT‐embryos. Mol. Reprod. Dev. 56:145–148, 2000. © 2000 Wiley‐Liss, Inc.