50 PRODUCTION OF THIRD-GENERATION CLONES OF A PIG BY SOMATIC CELL NUCLEAR TRANSFER

There has been a dominant view that serial cloning, i.e., cloning of a cloned animal, is only possible for a few generations. In this study, we examined the reproduction efficiency and normality of porcine offspring generated by serial somatic cell cloning. Salivary gland progenitor (SGP) cells were collected from a 4-month-old female cloned Landrace large white Duroc (LWD) pig (first generation, G1), which had been cloned from a fibroblast, and used as nuclear donors for second-generation clones (G2). The third generation of clones (G3) was produced by nuclear transfer using SGP cells from the G2 clones. Nuclear transfer was carried out by electric cell fusion using in vitro matured oocytes as recipients. Reconstructed embryos were electroactivated 1 to 1.5 hr after nuclear transfer, cultured for 1 to 2 days, and transplanted to the oviducts of estrus-synchronized surrogate gilts. A total of 391 embryos cloned from G1 animals were transplanted to three surrogates. All of the surrogates became pregnant and gave birth to a total of 13 (3.3%) of G2 clones (including two stillbirths). The average birth weight and size of eleven live piglets were 1203.6 � 113.5 g and 27.1 � 1.2 cm, both within the standard ranges of the original donor strain (LWD). Their growths until 8 months old were comparable to those of normal piglets of the same strain. For the generation of G3 clones, transplantation of 242 G2-derived embryos to two surrogate gilts resulted in one pregnant surrogate and three G3 clones (1.2%; average weight 1196.7 � 267.1 g and average size 35.7 � 2.3 cm), including a stillbirth. These results indicate that porcine serial cloning can efficiently generate up to three generations of apparently healthy clones, when SGP cells are used as nuclear donors. This study was supported by PROBRAIN.