Blastocyst Transfer

Blastocyst transfer is usually performed 24 hours after aggregation when the morulae have become expanded blastocysts and on the same day as injection. A little time is given between injection and transfer to allow blastocysts to re-expand.

The Recipient

Careful selection of the recipient is most important as the pups are the end result of a lot of hard work. Two strains of mice are used:RB Swiss and (CBundefinedC57BL6/J)f1. RB Swiss are quiet and make excellent mothers but they become overweight quickly and do exhibit bad planes of anaesthesia. CBundefinedC57 mice are hardy and display hybrid vigour. They do not carry excess weight and go under anaesthetic well. This strain can be very nervous when housed separately which could be dangerous to their young. They are most suitable if a young RB Swiss is placed into the cage as a companion which can be removed as soon as the pups are seven days old. By this age destruction of the litter is very unlikely. If the CBA mother is to be housed alone she must not be disturbed for ten days.

Prior to surgery, sterilise the following:
      - 3 pairs of curved forceps
      - 2 pairs of iris scissors
      - 1 pair suture clamps
      - 1 serafin clip
      - sterile suture with needle attached (small - for mouse surgery)
      - Michelle clips (small size)
      - Michelle clip applicators
      - 1 mouth pipette and flame-polished transfer pipette

You will also need an anaesthetic. Rompun / Ketavet is found to be quite effective. To make up 10ml:
      - 0.5ml 2% Rompun (20mg/ml Xylazine)
      - 0.5ml 100mg/ml Ketavet 100 - Delta Veterinary Lab, 8 Rosemead Rd Hornsby NSW 2077

Make up to 10ml with PBS

The dosage is 0.02 ml/g body weight

Store wrapped in tin foil at 4°C.

Shake well before use as it tends to separate in the fridge.

The Transfer

- Select a mouse that is 2.5 days pseudo pregnant and weigh. Do not use anything lighter and 25g or anything heavier than 30g. Underweight mice tend to re-absorb the embryos as they are not physically ready to support a pregnancy. Overweight mice make surgery difficult in the absorption of anaesthetic into the fat reduces the potency of the anaesthetic. 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.

- Anaesthetise mouse with Rompun / Ketavet, administered intraperitoneally. After administering the anaesthetic, put the mouse back into the box from which it came. The mouse will be more relaxed when placed in a familiar environment and the anaesthetic will act more quickly than it would on a distressed mouse.

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

- Take the anaesthetised mouse and shave its lower back. Lay mouse on 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 shaven area with hibitane or 70% ethanol.

- Instruments should have been laid out. Use one pair of iris scissors and one pair of forceps for cutting the skin - call these "outside" instruments. Use one pair of iris scissors and two pair of forceps for working inside the mouse - called the "inside" instruments.

- Using the outside forceps and scissors, make a small cut (about 1cm long)along the dorsal midline of the lower back. Through the shaven, moistened skin it is fairly easy to see blood vessels. Try to avoid these vessels when making the incision (see below).

- Using a pair of outside forceps and inside forceps, pick up the skin and separate skin from the body wall and cut the body wall as indicated about 5mm long, avoiding blood vessels.

- If the cut has been made in the right place, the ovarian fat pad is easily visible. If not, the fat pad can be located by lifting the edge of he body wall and scouting around with the other pair of inside forceps. Once you have located the fat pad attach serafin clip to it, taking care not to clip ovary. It is important not to damage the ovary as it is responsible for hormone production throughout pregnancy. Gently ease the ovary, oviduct and part of the uterus out through the incision in the body wall. DO NOT PULL. A traumatised uterus will contract and move quite violently, making surgery difficult and may cause expulsion of the transferred blastocysts. When the tip of the uterus is visible, rest the serafin clip across the mouse's back to hold the uterus in place. If the uterus or uterine horn continually slip back into the cavity it may be necessary to gently lie the mouse on the side being careful not to block the airway.

- The transfer pipette should now be loaded. Five or six embryos must be transferred to each horn, any less than this and the chances of a pregnancy resulting are serverely reduced. It may be loaded to suit yourself but this is a method that is popular. Take up a minute amount of DMEM with Hepes in the very tip of the transfer pipette, then make a small bubble by taking up a little air. Then take up some more medium - roughly the same volume as you hope to transfer the blastocysts in. Take up another bubble, same size as before. Then take up your blastocysts in the smallest possible volume of medium, lining them up side by side in the transfer pipette. This is how your transfer pipette should look when loaded.

- This will take some practise. Make sure that you are competent at loading a transfer pipette before any attempt at a blastocysts transfer. During surgery is not the time to learn how to load a pipette. If it is likely to take you more than a few minutes to load the transfer pipette, then do not expose the uterus until the pipette has been loaded. This prevents drying out and further trauma tot he uterus. Alternatively, the uterus, ovary, etc. may be moistened repeatedly with a sterile cotton bud and saline.

- Once the pipette is loaded and the uterus positioned, move the petri dish lid supporting the mouse to the microscope and turn on the overhead light source. Once the lights and focus have been adjusted and the mouse positioned to suit yourself, gently grasp the top of the uterine horn with a pair of inside forceps. Whilst still holding the horn with one hand, use the other hand to gently insert a 26 gauge hypodermic needle through the uterine wall (close to the oviduct) and into the lumen. Choose an area of the uterus that is relatively devoid of blood vessels as blood will clot in the tip of your pipette and block it. Remove the needle and carefully (so as not to lose the site of the hole), without averting your eyes, pick up the loaded transfer pipette. Gently insert the transfer pipette about 3mm into the uterine lumen. Gently blow the blastocysts into the uterus, using the air bubbles in your transfer pipette to monitor the transfer. Be careful not to blow any air into the uterus. Once transfer is complete, quickly rinse out transfer pipette in some HEPES buffer medium (M2) and check to see if there were any blastocysts stuck in the transfer pipette. If there were, transfer these blastocysts again.

- With the transfer complete, the serafin clip can now be removed and the uterus gently eased back into the body. Do not touch the uterus, but ease it back by the edges of the incision in the body wall and allowing the uterus to fall back in, without actually handling it. This procedure is then repeated on the other uterine horn. The incision in the body wall is not sutured. The skin is closed with Michelle clips - two per incision is usually sufficient. Michelle clips are used on the skin instead of suture as the mice will chew at the suture thread and effectively open their wounds up.

- Once surgery is complete, the mouse is placed in a box of clean autoclaved sawdust. Under anaesthetic, mammals are unable to retain heat as effectively as when conscious. For this reason, and also because the animal has been shaven, the mouse should be wrapped in a tissue to help keep it warm. This will also be used as bedding until the fur has grown back. It is also very important that the mouse by house alone as anaesthetised animals are often buried by cage mates who think they are dead. All animals should have recovered sufficiently from anaesthetic before being returned to the animal room and left unattended. The cage should be placed on a shelf away from male mice, as strange male pheromones will often cause females to abort. Recipient mice should be handled with care and tiptoed around as pregnant mice are easily upset, sometimes leading to abortion, or even cannibalism of pups.

If all goes well and a pregnancy results, the pups should be born sixteen days after the transfer.

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au

David Bowtell PMCI October 1998