Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells

• Published in: Molecular reproduction and development 2002-07, Vol.62 (3), p.300-306
• Main Authors: Lai, Liangxue, Park, Kwang-Wook, Cheong, Hee-Tae, Kühholzer, Birgit, Samuel, Melissa, Bonk, Aaron, Im, Gi-Sun, Rieke, August, Day, Billy N., Murphy, Clifton N., Carter, David B., Prather, Randall S.
• Format: Article
• Language: English
• Subjects: Animals; Animals, Genetically Modified; Biological and medical sciences; Biotechnology; Colchicine - pharmacology; Embryo Transfer; enhanced green fluorescent protein; Female; Fibroblasts - cytology; Fibroblasts - drug effects; Fundamental and applied biological sciences. Psychology; G2/M stage; Genetic engineering; Genetic technics; Green Fluorescent Proteins; Luminescent Proteins - genetics; Methods. Procedures. Technologies; nuclear transfer; Nuclear Transfer Techniques; Oocytes; pig; Swine - genetics; Transduction, Genetic; transgene; Transgenic animals; Transgenic animals and transgenic plants
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/mrd.10146

Fetal‐derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1‐cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2‐ to 4‐cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1‐cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine‐treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells. Mol. Reprod. Dev. 62: 300–306, 2002. © 2002 Wiley‐Liss, Inc.