Collecting Two-Cell-to Compacted Morula-stage Embryos

Materials

Animals

  • Pregnant female mice (20-60 hours p.c.) sacrificed humanely

Equipment

  • Embryo-handling pipette consisting of mouth or hand-held pipette assembly and  pulled capillary
  • Flushing needle (either a 30- or 32-gauge hypodermic needle [end cut and/or ground to a blunt tip on an abrasive stone (oilstone) or sandpaper]
  • Forceps, fine
  • Forceps, watchmaker’s #5, two pairs
  • Microdrop culture dish
  • Organ culture dish (Falcon 3037) (optional)
  • Petri dishes (35-mm) or embryological watch glasses
  • Scissors, fine
  • Stereomicroscope with transmitted and reflected or fiber optics (optional)
  • illumination (preferably a ground-glass stage) with 20x and 40x magnification.
  • Syringe, 1-cc

Reagents

  • Ethanol, 70%
  • M2 medium at room temperature

CAUTION:  See Appendix 2 for appropriate handling of materials marked with <!>.

Procedure

  1. To reduce the risk of tearing the oviduct, cut the end of a 30- or 32-gauge hypodermic needle and grind it to a blunt tip. It is also possible just to grind the sharp tip of the needle without cutting it to create a smaller beveled tip that may prove useful for flushing the oviducts from very young females.  Sterilize the needle by flushing it with 70% ethanol immediately before use.
  2. Open the abdominal cavity as described above.  Grasp the upper end of one of the uterine horns with fine forceps and gently pull the uterus, oviduct, ovary, and fat pad taut and away from the body cavity.  This will reveal a fine membrane (the mesometrium), which connects the reproductive tract to the body wall and carries a prominent blood vessel.  Poke a hole in the membrane close to the oviduct with the closed tips of a pair of fine forceps or scissors.
  3. Pull the oviduct, ovary, and fat pad taut with fine forceps and cut between the oviduct and ovary with fine scissors.  Do not be afraid to go close to the oviduct.  Reposition the forceps and cut the uterus near the oviduct, leaving at least 1 cm of the upper part of the uterus attached if the collection is taking place on 2.5 dpc.
  4. Transfer the oviduct and attached segment of uterus to a 35-mm Petri dish or embryological watch glass containing M2 medium at room temperature.  Oviducts from several mice can be collected in the same dish.  Place dish under stereomicroscope.
  5. Test the syringe to be sure that it is free of air bubbles and that the M2 medium is flowing smoothly before inserting the needle.
  6. The opening of the oviduct (infundibulum) at this time is no longer swollen and must be located within the coils of the oviduct.  Use fine forceps to slide the end of the oviduct onto the flushing needle.  Gently press the tip of the flushing needle against the bottom of the dish to hold it in place.  Flush the oviduct with ~0.1 ml of M2 medium.
  7. Use pipettes to pick up the embryos and wash them through several drops of fresh M2 medium to rinse off the debris.
  8. Transfer the embryos to a microdrop culture dish, rinse through several drops of equilibrated medium, and keep at 37ºC, 5% CO2 until needed.  An organ culture dish with equilibrated embryo culture medium may be used as an alternative for short-term incubation.

Procedure

  • To prevent the oviduct from moving while locating the infundibulum, it can be placed in a very small drop of medium or even onto dry plastic if it is moved directly from a drop of M2.
  • Because the tip of the flushing needle is blunt, it will not puncture the oviduct.  Therefore, it is often possible to use it as a tool to press down the oviduct to the plastic and hold it in place inside the infundibulum while flushing.
  • It is important to use only good-quality embryos for experiments and to distinguish them from delayed or fragmenting embryos.