Materials and equipment:
Sterile technique :
Autoclave 1 X PBS
Dissection tools (watchmaker forceps X 2 - 3, Surgical scissors)
Sterile dishes (10 cm)
Prepare 6 well dishes to culture the embryo cell culture and mark the well.
Prepare syringe contain required amount (300 ul) of trypsin and keep in hood.
Stopcock 3-way transfusion
Procedure:
1. Kill mice
2. Open abdomen and remove the both side of the uterus -- sterile from now!!
3. Open uterus and separate embryo (include decidua),
4. Take single embryo into fresh dish, open decidua, open and remove york sack for PCR genotype (soak in PBS of the 12 well dish, wash twice and dip into DNA buffer).
5. Rinse the forceps, take the embryo into a fresh syringe, carry the syringe contain the embryo into tissue culture hood. Place on a 3-way stopcock and add on a syringe containing 300ul trypsin on the other one side. Start timing before push the syringe, push the syringe from one side at a time, carefully close the stopcock bit by bit (not too much as the cells will be lysed), this requires about 20 seconds (shorter is better), and place syringe in one well then place dish on warm plate up to 3 minutes in total.
6. Rinse the syringe with 1 ml media and add total media of 3 - 3.5 ml. Do not use pipette take up and push down the culture since the embryo cells tend to be happier when the cells are aggregated.
7. Incubate the culture an 370C, 5% CO2 for 3-5 days till the cell reach to confluence, transfer the cell into 10 cm dish by 1 ml trypsin and 8 - 10 ml media