Biology Reference
In-Depth Information
4.
Place capillaries with 20-30
m-wide tips onto the grinding wheel at an angle of
about 20-45 degrees. Leave until a beveled end is formed.
ยต
5.
Connect the needle to a teflon tube linked to a syringe and wash it by sucking in
and out at least 3 times with the following solution: 10% hydrofluoric acid, DDW
1, DDW 2, DDW 3, 100% ethanol.
6.
Make a sharp fine tip at the beveled end. Heat the glass ball on the microforge
filament to a moderate temperature. Gently push the needle against the ball and
quickly withdraw it. The tip fuses with the glass ball and makes a sharp spike.
7.
Place the capillary orthogonal to the heating filament and with the opening toward
the operator. Bend the last 3-4 mm of the tip to form a 30 degree angle.
3.4.3. Mouse Matings
Blastocysts are obtained by natural mating of a relatively large number of
C57B6 mice. The presence of the vaginal plug the morning following mating
is considered 0.5 d postcoitum (dpc). Microinjected blastocysts are transferred
to the uteri of a pseudopregnant female. To help embryos recover from the
trauma of injection and to ensure a high rate of births, recipient females are one
day delayed in respect to blastocysts.
1.
Mate, at late afternoon, about 25 C576 males with 2 C57B6 females each (
see
Note 11
).
2.
The following morning check for plugs (
see
Note 5
) and keep pregnant females
in a separate cage.
3.
At late afternoon of the same day, mate vasectomized males with 2 CBA X C57B6
F1 females each.
4.
The following morning check for plugs (
see
Note 5
) and keep pseudopregnant
females in a separate cage.
3.4.4. Isolation of Blastocysts
1.
To isolate blastocysts, sacrifice by cervical dislocation C57B6 females at 3.5 dpc.
2.
Flush hair with 70% ethanol. Open the belly with scissors and expose the uterus
below bowels.
3.
Put the mouse under the stereomicroscope. Cut cervix (the single tube where
uterine horns join) with sharp, fine, curved scissors. Grasp the cervix with for-
ceps and gently lift the uterine horns. With fine curved scissors, cut mesometrium
and vessels along the uterine horns, and also being sure to avoid hurting the mus-
cular wall. Free the uterus by cutting at the utero-tubal junction. Place the uterus
in a drop of flushing medium.
4.
With forceps, hold a uterine horn near the utero-tubal junction. Using scissors
with the other hand, open the tip by making a cut parallel to the horn. Repeat this
procedure for the other horn. Place the uterus in a fresh drop of flushing medium.
5.
Place the uterus in a dry, sterile watch glass under the stereomicroscope. With
one hand, use forceps to hold the cervix. With the other hand, hold a 10-mL
syringe filled with flushing medium. Insert the needle in the cervix towards one
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