- Stem Cell & Developmental Biology
- Additive for ES | iPS
- 8 Cell Stage Embryo, Mouse (100embryos)
8 Cell Stage Embryo, Mouse (100embryos)
Cat. No. EE-001
Store at Liquid nitrogen
Producing eight-cell stage embryos
To obtain a maximum yield of eight-cell stage embryos from both SW and B6 strains, 3–4-week-old female mice were superovulated by hormone injection. Stud males are singly housed and are used twice weekly as breeders. Females are generated from our in-house colonies and are weaned at 21 days of age and either hormone injected immediately or housed for an additional week prior to use.
Pregnant mare's serum gonadotrophin (PMSG; Calbiochem) is intraperitoneally administered (5 IU/mouse) using a 1-ml syringe fitted with a 27.5-gauge needle at 5–6 pm approximately 48 h prior to mating. Human chorionic gonadotrophin (hCG; Calbiochem) is similarly administered (7.5 IU/mouse) 2 h prior to mating. To obtain a yield of about 150 eight-cell stage embryos at 2.5 days post coitum (dpc), thirty females are mated at approximately 4–5 pm. The females are visually inspected for a copulation plug the following morning (0.5 dpc). At 2.5 dpc (~ 9 am) the females are sacrificed by CO2 asphyxiation.
The mice are placed on a clean draping ventral side up. The abdominal fur is wiped with 70% ethanol. An incision is made along the ventral midline from the pubis to the rib cage. The skin and mesentery are resected and the viscera are moved aside to expose the ovaries, oviducts, and uterine horns. The oviduct is carefully cut away from the ovary and the uterine horn and flushed with ES media (− LIF) into a 35-mm culture dish using a 1-ml syringe fitted with a bent 30.5-gauge needle inserted in to the infundibulum.
After visual inspection under an MZ6 dissecting microscope (Leica), only the uncompacted eight-cell stage are selected for ES cell injection. To ensure the highest possible yield of fully ES cell-derived mice, the use of uncompacted eight-cell stage embryo is recommended. The embryos are placed into a new 35 mm dish containing a droplet of ES cell medium (− LIF) that has been overlaid with filtered mineral oil (no. M8410, Sigma-Aldrich). The embryos are incubated at 37 °C in a 7.5% CO2 atmosphere prior to use.