Biology Reference
In-Depth Information
8.
Using the holding pipet, gently suck a blastocyst. Moving the injection needle,
turn the blastocyst until a thin depression between two cells is in focus. Hold the
embryo in this position by delicate sucking (
Fig. 3
).
9.
By a rapid horizontal movement, insert the injection needle into the blastocyst
cavity. Inject about 15 cells. Gently withdraw the glass capillary and place
the injected blastocyst on the lower right corner. After injection, blastocysts
maycollapse.
10.
Repeat from
step 8
until cells are exhausted then repeat from
step 7
until all
blastocysts were used.
3.4.7. Embryo Transfer
After microinjection, blastocysts should recover in the incubator for at least
1 h. Generally, embryos can be transferred as soon as they start to reexpand to
form again their cavity. The number of transferred blastocysts for each
pseudopregnant female can vary from 7 to 12.
1. Anesthetize a pseudopregnant female mouse by intraperitoneal injection of
0.5 mL of diluted Avertin solution. Put the mouse with the head facing 12 o'clock
under the stereomicroscope equipped with optic fibers illuminators.
2.
Disinfect the skin of the back and cut with scissors at about 1-2 cm above the
hindleg. Wipe off hairs with a paper napkin. Detach the cut skin from the under-
lying muscle and displace it around to localize the underneath ovary (a reddish
ball surrounded by a white fat pad). Expose the ovary by cutting the above body
wall muscle, avoiding blood vessels (red) and nerves (white). Hold the fat with
blunt-ended curved forceps and gently force the uterine horn out of the cavity.
3.
On a second stereomicroscope, prepare the transfer pipet by filling it with a series
of small air bubbles followed by ES Medium. Cautiously suck 3-6 embryos,
minimizing the liquid in-between. Add a very small last air bubble at the tip.
Store the transfer pipet undisturbed nearby the first stereomicroscope.
4.
Using the fine forceps in one hand, hold the uterine horn near its apical end and
gently stretch it outside the abdominal cavity. With the other hand, quickly grab
the syringe needle and puncture the uterine wall avoiding the rupture of blood
vessels. Insert the needle so that it reaches the lumen of the uterine horn without
piercing again the musculature. Using this same hand, remove the needle and
take the mouth pipet, possibly avoiding to leave the binoculars. Locate the hole
again and insert the transfer pipet. Gently blow embryos until air bubbles are
seen to move inside the uterus. Slowly withdraw the pipet (
see
Note 13
). If liquid
is expelled from the hole, quickly suck it into the pipet (
see
Note 14
).
5.
Put the ovary and uterus back inside the abdomen. Using surgical catgut thread,
apply a few stitches to the muscle and to the skin.
6.
Repeat the same procedure from
step 2
on the other uterine horn.
7.
When finished, put the mouse back to the cage and keep it warm by covering it
with straw.
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