Culture of Embryos
After fusion/activation, 20-30 reconstructed embryos were transferred to a 50-|xl drop of culture medium covered with mineral oil in a 35-mm dish, and the dishes were held in 5% CO2 in air at 39°C. Nonmanipulated oocytes were electrically activated and cultured as controls. Some reconstructed embryos were stained with 5 |xg/ml bisbenzimide (Hoechst 33342) to identify nuclei by using an epifluorescent microscope (Nikon, Japan). After 6 days of culture all embryos were stained with Hoechst 33342 to determine the number of nuclei by using an epifluorescent microscope, and embryos with two or more nuclei were determined to have cleaved. To detect eGFP expression, embryos were examined on an epi-fluorescent microscope using a standard fluorescein isothiocyanate (FITC) filter set.
To eliminate any potential of a culture-induced detrimental affect on development, embryos (1- to 2-cell stage) that had been cultured for 3-5 h or for 1 day (or both) after fusion were surgically transferred into one oviduct of each gilt. Pregnancy status was monitored by using an ultrasound scanner.
Polymerase Chain Reaction Analysis for the EGFP Gene
Genomic ear skin DNA from four NT piglets, a donor pig, and the surrogate mother was extracted and subjected to polymerase chain reaction (PCR) analysis using two sets of primers. For the eGFP gene, the eGFP forward primer (5′-CGCACCATCTTCTTCAAGGACGAC-3′) and the reverse primer (5′ -AACTCCAGCAGGACCATGTGATCG-3′) were used. A 383-base pair (bp) amplicon was generated after eGFP gene amplification. PCR consisted of 32 cycles at 94°C for 45 sec, 61 °C for 30 sec, and 72°C for 45 sec. PCR products were run on a 1% agarose gel.