Embryo transfer

Ovulation was induced in A/J female mice via the intraperitoneal administration of 5 IU PMSG between 12:00 and 2:00 p.m. Two days later, 5 IU of hCG was injected intraperitoneally after random mating with A/J male mice. At 0.5 days post-coitum (d.p.c.), female mice with a copulatory plug were separated from male mice. At 1.5 d.p.c., female A/J mice were euthanized via cervical dislocation. The abdominal cavity was opened, and the oviducts were aseptically collected. Embryos were flushed from the oviducts using M2 medium (supplemented with Pen-Strep 100x) and collected in a pool with a mouth transfer capillary setup under a stereoscopic microscope (SMZ-10, Nikon). Two-cell embryos with intact zona pellucida were washed 10 times in M2 medium. Recipients consisted of nulliparous C56BL/6 female mice. Each female was mated with a vasectomized male mice C57BL/6 near the end of the light cycle period. Embryo transfers were performed at 0.5 d.p.c. in recipient females with a copulatory plug. Surgeries were performed within the SPF facility clean area under a horizontal laminar flow cabinet. Females were anesthetized with 0.2% acepromazine (2 mg/kg), 10% ketamine hydrochloride (100 mg/kg), and 2% xylazine hydrochloride (10 mg/kg) via intraperitoneal injection. A sterile saline solution was used to prevent corneal drying during the surgery. Each female was placed in sternal recumbency on a digital heating plate at 37 °C. The peritoneal cavity was accessed via a musculature incision over the ovary fat pad. Under stereoscopic microscopy (SMZ 2-B, Nikon), a small incision was made in the bursa between the ovary and the oviduct to expose the infundibulum. Each female received 20 embryos equally that were divided between the oviducts using a glass micropipette. Pups were born in 3 weeks and weaned after 21 days.