Transgenic Research BW2118: 1–10, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 1 Viable transgenic goats derived from skin cells Esmail Behboodi, Erdogan Memili, David T. Melican, Margaret M. Destrempes, Susan A. Overton, Jennifer L. Williams, Peter A. Flanagan, Robin E. Butler, Hetty Liem, Li How Chen, Harry M. Meade, William G. Gavin & Yann Echelard ∗ GTC-Biotherapeutics Inc., 5 Mountain Road, Framingham, MA, 01701, USA Received 1 September 2003; accepted 24 December 2003 Key words: cell cycle, cyclin D 1 , embryo, goat, nuclear transfer, transgenic Abstract The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G 0 ) by serum starvation and then induced to exit G 0 and pro- ceed into G 1 . Oocytes collected from superovulated donors were enucleated, karyoplast–cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25–30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2–3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line. Introduction Recent advances in the large scale production of hu- man recombinant proteins have had a significant im- pact on the pharmaceutical industry. The mammary gland of large animals is well suited for production of these proteins (Clark et al., 1998; Meade et al., 1998). Therapeutic proteins, such as alpha-1 antitryp- sin, antithrombin, and several monoclonal antibodies (Wright et al., 1991; Edmunds et al., 1998; Pollock et al., 1999) have been produced in the milk of trans- genic animals. Among the benefits of this technology are high production yields, low capital investment, and the elimination of reliance on products derived from human blood. ∗ Author for correspondence E-mail: yann.echelard@gtc-bio.com Until recently, the only reliable method available for producing transgenic farm animals has been pro- nuclear microinjection. The success rate of this tech- nique has been low, with 0.5–3% of microinjected embryos giving rise to transgenic offspring (Hammer et al., 1985; Bondioli et al., 1991; Ebert et al., 1994; Gavin, 1996; Behboodi et al., 2001). The emerging use of transfected cultured somatic cells as karyoplast donors for nuclear transfer (NT) has several advan- tages over microinjection, and has facilitated the gen- eration of transgenic animals (Campbell et al., 1996; Wilmut et al., 1997; Cibelli et al., 1998; Baguisi et al., 1999; Onishi et al., 2000; Polejaeva et al., 2000). NT using transfected somatic cells allows the prescreen- ing of cells for desirable genotypic characteristics which can reduce the number of animals (donors and recipients) used during the production of transgenic animals. In cattle, sheep and goats, both fetal and adult bw-2118.tex; 20/02/2004; 10:35; p.1 Thomson Press (I) Ltd., PIPS No. 5267172 (tragkap:bio2fam) v.1.2 Uncorrected proof! Pdf output