Making New ES Lines

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Dissection of 3.5dpc mouse embryos

4-5 plugged females should be delivered to the investigator's lab on Thursday morning.

Prior to dissecting embryos prepare the following:

(5) Take a bunny suit, mask, gloves, and appropriate tools with you to the investigator's lab. Gown up.

(6) Using a separate set of tools, dissect out the uteri from all the females, one at a time. {Make these dissections as clean as possible: the less fat and debris, the easier it will be to see the embryos after you flush them out}

(7) Place the uteri into a large drop of FHM or M2 medium in a 35mm dish. Discard gown and return to transgenic lab.

(8) Back in the transgenic lab, put on a new gown, mask, and gloves. Flush out the embryos in a surgery hood with dissecting microscope.

(9) Using separate tools, cut a single half of a uterus just above the cervix. Place it dry into a clean 35mm dish. Visualize the infundibulum and flush with 0.1-0.2ml of medium toward the cut end of the uterus.

(10) Remove the flushed half of a uterus and visualize embryos under the dissecting microscope. Collect embryos and place in the collection plate (drop1).

(11) Continue with all the other halves of uteri. Collect all embryos into drop1 of the collection plate.

(12) Gently transfer the embryos through the other 3 drops on the collection plate. All the embryos will be together in drop4.

(13) Discard gown and transfer the collection plate into the tissue culture lab.

(14) Visualize embryos under the tissue culture microscope (4x objective). Remove appropriate number of 4-well PMEF plates from incubator (each well with 1ml of SR-medium).

(15) Transfer an individual embryo to an individual well. Number all wells that contain an embryo. Return all plates to the cell culture incubator and leave undisturbed for 6 days. {Count the Thursday of culture as Day 0}

(16) The goal is 25-40 embryos. Depending on the yield of embryos from the first collection, contact the investigator’s lab if you need them to go through another cycle of breeding. Mating, preparation of PMEF plates, dissection, and collection of embryos will be repeated exactly as previously described. After two cycles of breeding, stop. Do not continue embryo collection until the ES cell derivation procedure has been completed.


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