In vitro developmental ability was determined after 6 days in vitro. The blastocyst formation percentage of control parthenogenetic embryos (26.1% ± 2.7%) was higher (P < 0.05) than either NT group (9.4% ± 1.5% and 10.0% ± 1.2%). The mean cell number of blastocysts (28.8 ± 2.8 to 33.5 ± 2.2) was not different (P > 0.05) among control and NT groups. In the NT-fetal fibroblast (NT-FF) group all embryos expressed fluorescence, but in the NT-ear skin fibroblast (NT-EF) group only 26.4% of cleaved embryos expressed green fluorescence. Among embryos expressing eGFP, 58.9% appeared mosaic in the NT-eF group (Table 1, Fig. 2).
NT-EF embryos were surgically transferred to five surrogate gilts, and one gilt delivered four female piglets (NT7 to NT10) on Day 113 of gestation by caesarian method (Table 2). Birth weights ranged from 850 g to 1300 g, and placental weights ranged from 189 g to 374 g. Microsatellite analysis by using three markers suggested that all four piglets were derived from the ear skin fibroblast cell line (Table 3). PCR results confirmed the presence of the eGFP gene in all piglets and the donor cell line (Fig. 3). All four gilts expressed eGFP, as did the original donor. All had coloration of the snout, oral and hoof wall of each limb as the former report. NT8 died at 3 days of age from a bacterial infection, and NT9 died at 7 days of age due to congestive heart failure.
FIG. 1. Expression of eGFP in ear skin fibroblasts (EFs). A) EFs under normal light. A’) EFs under an FITC filter set.
FIG. 2. Expression of eGFP in NT embryos derived from ear skin fibroblasts. A-F) Embryos stained with Hoechst 33342 under UV light to identify the nuclei number. A’-F’) Embryos under FITC filters to identify eGFP expression. A-B’) Four-cell stage embryos. C-D’) Eight-cell stage embryos. E-F’) Blastocyst stage embryos. A’, C’, and E’) Whole eGFP expression. B’, D’, and F’) Mosaic eGFP expression.
FIG. 4. Two NT piglets (NT7 and NT10) derived from ear skin fibroblasts of the transgenic pig. NT7 had abnormal feet. One of the dewclaws of the front left leg was abnormally large (short arrow), and the front right leg had three dewclaws (long arrow shows additional dewclaw).