*4.5. Blastocyst Collection and Transfer*

All animal procedures were approved by Institutional Animal Care and Use Committee at Colorado State University, Fort Collins, Colorado, USA. Blastocysts collection and transfer was done following the procedure previously described by Baker at al [61]. A group of 12 ewes at day 6-12 of estrus were synchronized by two intramuscular injections of prostaglandin F-2α (PGF-2α) at 10 mg/dose given at interval of 4 h (Lutalyse, Pfizer, New York, NY). After 48 h of estrus synchronization, 4 ewes were separated to be used as recipients while 8 donor ewes were bred by intact rams. At day 9, donor ewes were euthanized using pentobarbital sodium (90 mg/kg IV, Pentasol, Vibrac, Fort Worth, TX, USA), and blastocysts were flushed from the uterus using DMEM-F-12 (1:1) medium supplemented with 0.25% BSA. The hatched blastocysts were infected with 100,000 shRNA expressing lentiviral particles in a 100 µL drop of CDM-2 media with 5 µg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). Blastocysts with lentiviral particles were kept in incubator for 4–5 h at 5% CO2, 5% O2, and 38.5 ◦C. Overnight-fasted recipient ewes were sedated using ketamine (12.5 mg/kg IV, Ketacine, VetOne, Boise, ID, USA) and diazepam (0.125 mg/kg IV, Hospira, Lake Forest, IL, USA). Surgical procedure was performed under general anesthesia on 2 L/min O2 and 2–4% isoflurane (Fluriso, VetOne, Boise, IS, USA). A total of 23 blastocysts were transferred including 6 SC, 7 AKD, and 10 BKD. One blastocyst was transferred in each recipient.
