Biology Reference
In-Depth Information
3.
Electroporate 10
7
ES cells as described in
Subheading 3.3.2.
Plate about 5
×
10
6
ES cells/9-cm dish seeded with feeder cells.
4.
The appearance of colonies of mixed genotype (floxed and deleted) is avoided by
trypsinizing cell after the minimum amount of time required for
Cre
to exert its
function: as soon as colonies appear (usually 2-3 d after transfection) trypsinize
plates and reseed about 1/3 of the cells on the same dish. Add irradiated embry-
onic fibroblasts so that the feeder layer is always confluent. In case a
neo-tk
selection cassette is flanked by
loxP
sites, negative selection of deleted clones
can be applied by adding 2
µ
M
Gancyclovir to the medium.
5.
As soon as colonies appear again (3-4 d after
step 4
) proceed to pick and freeze
as described in
Subheading 3.3.
6.
Identify the clones bearing the desired deletion by Southern blot as described in
Subheading 3.6.4.
3.4. Generation of Mutant Mouse Lines
Chimeric mice are generated by microinjection of mutant ES cells into the
cavity of a blastocyst
(33)
. Chimeras are then mated to generate an offspring
that carries the ES cells genotype. To establish these techniques, an animal
care facility must be available and treatment of mice must proceed in agree-
ment to local laws regulating in vivo experimentation.
3.4.1. Generation of Vasectomized Males
1.
Anesthetize a male mouse by intraperitoneal injection of 0.5 mL of diluted
Avertin solution. Wash skin of lower abdomen (at the level of the top of the legs)
with 75% ethanol and make a 1-cm cut with sharp scissors (1.5-cm large). Simi-
larly cut the muscle of the body wall, avoiding the fat pad surrounding the genitals.
2.
With bent blunt forceps, gently push the scrotum to move the right testicle into
the abdominal cavity (until the white testicular fat pad appears at the edge of the
incision). Expose the testicle by pulling the white fat pad. Note that around the
testicle, the white coiled epididymis prolongs in a wider tube: the vas deferens.
3.
Pierce with the tip of the forceps the thin membrane linking the vas deferens to
the testicle and blood vessel. Apply two stitches around the freed tube, at a dis-
tance of about 5 mm from each other. With scissors, cut the vas deferens between
the two stitches. By gently grasping the fat with forceps, reposition the testicle
inside the abdomen. The same procedure is repeated for the left testicle.
4.
Separately stitch 2 or 3 times muscle and skin, then put the mouse alone back into
a fresh cage. Vasectomized mice can be used 15-30 d after surgery. Testing for
residual breeding capacity is advisable.
3.4.2. Preparation of Needles for Microinjection
Two needles are needed: one is used to hold the blastocyst and the other to
suck and inject cells (
Fig. 3
). Making such microinjection needles is a labori-
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