This study aimed at collecting background knowledge for chimeric pig production. We analyzed the genetic sex of the chimeric pigs in relation to phenotypic sex as well as to functional germ cell formation. Chimeric pigs were produced by injecting Day 6 or Day 7 inner cell mass (ICM) cells into Day 6 blastocysts. Approximately 20% of the piglets born from the injected blastocysts showed overt coat color chimerism regardless of the embryonic stage of donor cells. The male:female sex ratio was 7:2 and 6:1 in the chimeras derived from Day 6 and Day 7 ICM cells, respectively, showing an obvious bias toward males. When XX donor cells were injected into XY blastocysts at the same embryonic stage, the phenotypic sex of the resulting chimera was male with no germ-line cells formed from the donor cell lineage. On the other hand, when the donor was XY and the recipient blastocyst was XX, the phenotypic sex of the chimera was male, and germ-line cells were derived only from the donor cells. The combination of XY donor cells and XY blastocysts produced some chimeras in which the donor cell lineage did not contribute to germ-line formation even when it appeared in coat color. When the embryonic stage of the donor was advanced by 1 day in the XY-XY combination, 100% of the germ-line cells of the chimeras were derived from the donor cell lineage. These data showed that characteristics of sex differentiation and germ cell formation in chimeric pigs are similar to those in chimeric mice.

We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.