Generating Gene Knockout Mice for Studying Mechanisms Underlying Drug Addiction - Drugs of Abuse: Neurological Reviews and Protocols - page 351

loxane. Kill all plugged female C57BL/6J mice by cervical dislocation. Dissect
out uteri and fl ush into a 50-mm Petri dish with ES medium using the syringe.
Collect blastocysts under the microscope using a transfer pipet connected to a
mouthpiece. Transfer all blastocysts into the above culture dish and leave the
dish in the tissue culture incubator. Although the yield may vary, 50 blastocysts
typically can be obtained.
3.3.3. Blastocyst Injection
1. Setting up an injection dish: In the center of a 50-mm Petri dish, place 0.5 mL of
ES medium in a rectangular way. Cover the medium with dimethylpolysiloxane.
Transfer about 10 blastocysts and a few hundred ES cells into the injection dish
by mouth pipeting. Adjust both the holding pipet and the injection pipet so that
they are in the same plane.
2. Blastocyst injection: Use the injection pipet to collect undifferentiated ES cells
with minimum volume of medium under the microscope. Avoid air bubbles.
Find and hold a healthy blastocyst from the side with the inner cell mass using
the holding pipet. Move the tip of the injection pipet close to the blastocyst
and penetrate the wall of the blastocyst. Gently release 8-10 ES cells into the
blastocyst. Gently move the injection pipet when exiting the injected blastocyst
to prevent the ES cells from being expelled out. Repeat the injection process
( see Note 9 ).
3. Blastocyst culture: Transfer the injected blastocysts to another Petri dish contain-
ing the ES medium covered with dimethylpolysiloxane. Culture the injected
blastocysts in the incubator for at least 2 h before returning them to foster
mothers.
4. Additional injections: Each ES clone should be injected at least twice. Moreover,
three or four independent ES clones should be injected ( see Note 10 ).
3.3.4. Transfer of the Injected Blastocysts Back into Foster Mothers
1. Anesthetize the foster mothers by an intraperitoneal injection of 2.5% avertin
at 0.016 mL/g of body weight.
2. Make a small incision on one side of the mouse and avoid lesioning blood
vessels. Pull out one side of the uterus through the associated fat using a pair
of forceps.
3. Collect about six to eight injected and two noninjected blastocysts using a
transfer pipet connected to a mouthpiece. Wash the blastocysts twice in fresh
ES cell medium.
4. Gently punch the uterine wall from the oviduct end of the foster mother with a
25-gauge needle. Then, put the tip of the transfer pipet in and gently blow the
blastocysts into the uterus. Close the incision with a wound clip.
5. Leave the foster mothers in a cage on top of a 37°C warm plate till waking up.
Return foster mothers to the mouse colony. Single house foster mothers when
they are ready to give birth.