Biology Reference
In-Depth Information
horn. Flush with about 0.5-1 ml, tightening the muscular wall around the needle
with forceps. If the uterine tube does not let liquid out, repeat the procedure in
step 4.
6.
Collect blastocysts under the stereomicroscope by sucking them into a transfer
pipet. Transfer blastocysts in a watch glass containing ES Medium and keep them
in the incubator until needed
3.4.5. Preparation of ES Cells
1.
Plate ES cells and feeder on a 6-cm large tissue-culture dish 2-3 d before micro-
injection. Change ES Medium every day. ES cell colonies should reach an opti-
mal density (75-90% confluency) without being passaged.
2.
C.
3. Add 3 mL ES Medium. Carefully dissociate colonies by pipeting up and down.
Transfer the cell suspension to a 10-mL sterile tube. Spin 5 min at 1000 RPM.
4. Resuspend pellet in 5 mL ES Medium. Discard about 4 mL and spin the rest
5 min at 120
g
.
Wash cells 2 times with 5 mL PBS. Add 0.5 mL trypsin. Incubate 5 min at 37
°
5.
Discard the supernatant leaving just a drop medium. Resuspend cells by gently
flickering the tube with fingers.
3.4.6. Microinjection of ES Cells
Blastocyst injection requires an inverted microscope equipped with phase
contrast or Nomarski's optics. Two micromanipulators are set at the left and
right sides of the microscope's stage. Each micromanipulator controls move-
ments of a glass capillary. One needle (on the left) is used to hold the blasto-
cyst and the other (on the right) to collect and inject cells. Each capillary is
held through a hollow metal rod connected via a silicon tubing to an air filled
syringe. Sucking and blowing of cells and embryos is controlled only by a
gentle action on this device.
1.
With vaseline grease, fix a siliconized coverslip to the injection chamber.
2.
Put a large drop of M2 medium on the coverslip and, using the transfer pipet, add
blastocysts at the upper left corner. In a similar way, add the suspension of ES
cells in stripes along the drop.
3.
Cool the stage of the microscope to about 10
C by placing on its top a glass Petri
dish filled with ice. Put the injection chamber on the cooled stage.
°
4.
Connect capillaries to their metal holder and syringe. Fill the pipets with M2
medium (
see
Note 12
).
5.
Adjust holding and injection pipets on micromanipulators. Put capillaries in focus.
Pipets should be carefully turned until they show a straight horizontal orientation.
6.
Overlay the M2 drop with dimethyl polysiloxan.
7.
Collect about 150 healthy ES cells. Healthy ES cells are round and of medium
size. They must have a smooth surface and a bright nucleus. Feeder cells can be
easily avoided by their larger size and spiked shape (
Fig. 3
).
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