Preparation of Blastocyst Stage Embryos from Pseudopregnant Mice

Approximately 4 days after the presence of vaginal plugs, the blastocysts usually present in the uterine lumen can be removed by flushing the uterine horns with embryo culture medium. Surgical gloves should be worn during this procedure.

1. Kill the pregnant females on the fourth day (84 to 96 h) of pregnancy by cervical dislocation.

2. Briefly clean the abdomen with 70% ethanol and make a small, transverse incision in the middle area of the abdomen. Remove the cut hairs with alcohol-soaked cotton.

3. At the incision site, tear the skin and pull it away to the front and hind legs, respectively. At this point, the peritoneum should be exposed.

4. Make a large, transverse incision in the peritoneum to expose the abdominal cavity.

5. Identify the reproductive tract by pushing aside the intestines and use blunt forceps to grasp the point of bifurcation of the uterus near the base of the bladder.

6. Carefully lift the uterus with blunt forceps, remove the supporting mesentery on both sides of the uterus, and cut across the cervix at the base.

7. Remove the entire tract by cutting it between the oviduct and the ovary and place it in a sterile Petri dish.

8. Under the low power of a dissecting microscope, carefully remove any mesentery and fat. At the utero-tubal junction site, cut off the oviduct and make an approximately 0.5 cm longitudinal clip at the end of each horn. Finally, cut across the cervix, exposing the entrances to both uterine horns.

9. Rinse the tract with embryo culture medium and place it in a sterile Petri dish. Take up 0.5 ml of embryo culture medium using a small Pasteur pipette or a pipette attached to a sterile 1-ml tip, insert the tip into the cervical end and gently flush the lumen a few times. The uterus should be slightly inflated and the medium containing embryos should flow through the tract.

10. Under the low power of a dissecting microscope, quickly transfer the blastocyst-stage embryos that have settled to the bottom of the flush dish into a micromanipulation chamber or dish using a sterile Pasteur pipette or pipette tip.

Materials Needed

Small blunt, curved forceps Watchmaker's forceps Small sharp scissors Iridectomy scissors Surgical gloves 70% ethanol and cotton

Embryo Culture Medium (1 l)

Na2HPO412H2O, 3.0 g NaCl, 6.2 g KCl, 0.2 g KH2PO4, 0.2 g CaCl2.2H2O, 0.1 g MgCl2, 0.1 g Na pyruvate, 0.05 g Penicillin, 62 mg D-Glucose, 1.04 g Phenol red, 100 mg

Dissolve well after each addition in 1000 ml distilled water. Add 10% (v/v) of heat-inactivated fetal calf serum to the medium. Filter-sterilize and aliquot prior to use. The medium can be stored at 4°C for up to 5 weeks.

Preparation of Micromanipulation Apparatus

Micromanipulation can be set up with a micromanipulator system, including (a) an inverted microscope or a standard microscope (Leitz Instruments, Ltd.); (b) two sets of micromanipulators (Leitz Instruments Ltd.); (c) Kopf needle puller model 750 (David Kopf Instruments); (d) a finer needle and a pipette holder; and (e) a Schott-KL-1500 cold light source (Schott).

1. Holding pipette: To hold and stabilize a blastocyst during the injection of ES cells, a holding pipette needs to be prepared as follows:

a. Pull out a length of glass capillary with a pipette puller according to the manufacturer's instructions or by hand over a low flame.

b. Break the pipette at the point of 70 to 90 mm with a microforge according to the manufacturer's instructions. Fire-polish the tip of the pipette and close it down to a diameter of approximately 15 mm. The pipette may be further bent so that it can be placed horizontally in the microscope field.

2. ES cell injection pipettes: These pipettes will be used to inject ES cells into the blastocyst. The procedures are similar to those described for the holding pipette except that the tip should be broken off to generate about 12 to 15 mm for ES cells to pass through. No fire-polishing and bending are necessary.

3. Blastocyst injection pipettes: This type of pipette will be utilized to transfer blastocyst embryos for injection of ES cells into the embryos. The procedures are similar to those described for holding pipettes except that the tip should be broken off to generate a relatively large inside diameter (about 130 mm) for the blastocyst to pass through and fire-polishing should be very brief to eliminate the sharp edges.

4. Embryo transfer pipettes: These pipettes will be employed to transfer the blastocyst embryos containing the injected ES cells back to the uterus of the host. The procedures are similar to those described for holding pipettes except that the tip should be broken off to generate a relatively large inside diameter (about 130 mm) for the blastocyst to pass through freely and fire-polishing should be very brief to eliminate the sharp edges.

Injection of ES Cells into Blastocysts

1. Set up a suitable schedule so that ES cells may be trypsinized and gently suspend them to maintain them as a single cell suspension in embryo culture medium at 4°C prior to use; the blastocysts may be prepared in the morning.

2. Take up a tiny volume of light liquid paraffin (optional) by positive pressure and then take up blastocysts or ES cells into an embryo transfer pipette by capillary suction. Under the microscope, introduce the blasto-cysts into drops of embryo culture medium in a chamber, dish or depression slide.

3. Assemble all parts necessary for the injection according to the manufacturer's instructions.

4. Position the pipette tips so that they are parallel in the field using the coarse adjustments of the manipulator. The holding pipette and ES cell injection pipette should be placed in opposite directions (e.g., north and south). Focus and place them in the same plane, utilizing the fine adjustments.

5. Carefully pull the pipettes far enough out to allow the introduction of a chamber, dish or slide containing the blastocysts onto the stage.

6. Carefully reposition the pipettes and focus. Position and place the relatively dark side of the blastocysts in the center of the field, then carefully bring the ES cell pipette closer to the blastocyst.

7. Carefully insert the ES cell pipette and expel one to five ES cells into the blastocoelic cavity (relatively light part of the blastocyst). Slightly remove the ES cell pipette and put the injected blastocyst aside.

8. Repeat the procedure for other blastocysts.

Reimplantation of Injected Blastocysts into Uterus of Recipient Female

1. Prepare the third- or fourth-day pseudopregnant females as described previously. It is recommended that these mice be prepared 1 day later than the mice prepared as the blastocyst donors.

2. Weigh and anaesthetize a couple of females as described earlier.

3. Clean the back of a female with 70% ethanol and make an approximately 1-cm transverse incision in the skin in the area of the first lumbar vertebra. Clean the cut hairs with cotton wetted in alcohol.

4. Carefully slide the skin incision to both sides until the left ovarian fat pad and the ovary can be seen through the peritoneal wall.

5. Make a 4-mm incision through the peritoneum. Locate the ovary, oviduct and uterus using blunt forceps. Grasp the fat pad and carefully pull it out together with the uterus through the opening.

6. Under a dissecting microscope, hold a pipette loaded with the embryos and a hypodermic needle in the right hand, and carefully hold the uterus with forceps in the left hand. Insert the needle through the muscle layers of the uterus and position the needle parallel to the long axis of the uterus in order to allow the tip to enter the lumen.

7. Pull out the needle and insert the tip of the embryo transfer pipette through the hole made by the needle into the lumen of the uterus. Carefully expel all of the blastocysts into the lumen and remove the pipette. Bubbles should be avoided as much as possible.

8. Replace the uterus and perform the same procedure for the other side if bilateral transfer is necessary.

9. Seal the incision or close it with a small wound clip.

10. Recover the foster mothers by briefly warming them under an infrared lamp (do not overheat), and house two or three in one cage. Live offspring are usually born 18 days after the surgery. The initial offspring are called the founder or GO animals in terms of genetics. The offspring can be allowed to breed to maintain the animals.

Method B. Production of Transgenic Mice from Oocytes

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