Blastocyst transfer is best performed after allowing injected embryos a little recovery time in culture. This allows better evaluation of the cells' survival - the embryos should recavitate if they have not been damaged during injection.

The Recipient

Careful selection of the recipient is most important as the pups are the end result of a lot of hard work. I personally use Swiss Webster or CD-1 mice, as they are quiet and make excellent mothers, although do become overweight quickly and exhibit bad planes of anesthesia when heavy. This is also a very inexpensive mouse to use. As an alternate, another strain I have used with considerable success is B6D2F1. These mice are hardy and display hybrid vigor.

The Equipment

For surgery, I clean (with 70% alcohol) the following (all Roboz instruments):

5 pairs of forceps       1 pair Pattern 3c       1 pair straight serrated, 3.5"       1 pair curved serrated, 3.5"       1 pair of 3.5" sharp / sharp scissors (Surecut) 

25 guage hypodermic needle

Autoclip metal wound clipper

1 serafin clip

1 mouth pipetter and hand-pulled transfer pipette

The Anesthetic

Avertin (2,2,2 tribromoethanol) is found to be quite effective. For method of preparation, see "Manipulating the Mouse Embryo", CSHL Press, ISBN 0-87969-384-3. Store wrapped in tin foil at 4oC as this reagent is light sensitive. Test after making a new batch. Shake well before use.

The Transfer

Select a mouse that has been plugged by a vasectomized male 2.5 days previously. Do not use an mouse that appears lighter than 25g, as underweight mice tend to re-absorb the embryos as they are not physically ready to support a pregnancy. Overweight mice can make surgery difficult by the absorption of anaesthetic into the fat reducing the potency of the anesthetic; also, the presence of fat means the presence of blood vessels, and cutting through all the extra fat causes a lot of unnecessary bleeding. This makes it difficult to see what you are doing and may also clog up the tip of your transfer pipette.

Anesthetise mouse with Avertin, administered intraperitoneally. After administering the anesthetic, put the mouse back into the cage from which it came. The mouse will be more relaxed when placed in a familiar environment and the anesthetic will act more quickly than it would on a distressed mouse.

To check that the mouse is fully anesthetised, press or squeeze the pads of the feet. If the mouse can feel this it will try to withdraw its leg from your grasp (Pedal reflex). Do not commence surgery until there is no reflex reaction to this test.

Take the anesthetised mouse and lay it on it's belly on a petri dish lid, taking care to keep the airway clear by resting the teeth on the edge of the petri dish. This makes it easier to move the mouse around without having to actually touch it. Swab the incision area with 70% ethanol.