Oocytes were processed as previously described [5] (link). Pig ovaries were purchased from a local slaughter house. Briefly, cumulus oocyte complexes (COCs) were aspirated from antral follicles and washed with maturation medium (Tissue Culture Medium 199) (Gibco) supplemented with 0.1% (w/v) polyvinyl alcohol (Sigma), 3.05 mM d -glucose, 0.91 mM sodium pyruvate (Sigma), 0.57 nM cysteine (Sigma), 0.5 µg/ml LH (Sigma), 0.5 µg /ml FSH (Sigma), 10 ng/ml epidermal growth factor (Sigma), 10% (v/v) porcine follicular fluid, 75 µg /ml penicillin G, and 50 µg /ml streptomycin. The COCs were then transferred to a four-well multidish (Nunc) containing 500 µl of the maturation medium covered with mineral oil and pre-equilibrated at 39°C overnight. After 42–44 h of incubation, the oocytes were released from the COCs by vigorous vortex for 4 min in TL-Hepes containing 0.1% polyvinyl alcohol and 0.1% hyaluronidase (Sigma). The polar body and associated metaphase plate of the oocytes were aspirated using glass pipettes in micromanipulation medium supplemented with 7.5 µg/ml cytochalasin B. Denuded oocytes were used as the recipients for SCNT.
The transgenic PFFs were thawed and grown to subconfluency prior to SCNT. Cells with uniform co-expression of the four fluorescent proteins were selected under fluorescent microscopy and used as donor cells that were injected into the perivitelline space of the oocytes. The oocyte-donor cell complexes were activated to fuse and become reconstructed embryos with two successive DC pulses at 1.2 kv/cm for 30 µs using an electrofusion instrument (BLS). The reconstructed embryos were cultured in embryo-development medium PZM3 at 39°C overnight and then surgically transferred to recipient gilts exhibiting natural estrus. The pregnancy status of the gilts was monitored weekly using an ultrasound scanner beginning on Day 24 after the implantation. All cloned piglets were delivered by natural birth. To monitor in vitro development, transgenic and nontransgenic SCNT embryos were cultured for 6 days until they reached the blastocyst stage.
The transgenic PFFs were thawed and grown to subconfluency prior to SCNT. Cells with uniform co-expression of the four fluorescent proteins were selected under fluorescent microscopy and used as donor cells that were injected into the perivitelline space of the oocytes. The oocyte-donor cell complexes were activated to fuse and become reconstructed embryos with two successive DC pulses at 1.2 kv/cm for 30 µs using an electrofusion instrument (BLS). The reconstructed embryos were cultured in embryo-development medium PZM3 at 39°C overnight and then surgically transferred to recipient gilts exhibiting natural estrus. The pregnancy status of the gilts was monitored weekly using an ultrasound scanner beginning on Day 24 after the implantation. All cloned piglets were delivered by natural birth. To monitor in vitro development, transgenic and nontransgenic SCNT embryos were cultured for 6 days until they reached the blastocyst stage.
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