Egg-transfer pipets

Two types of egg-transfer pipets need to be prepared in advance general transfer pipets and oviduct-transfer pipets. These are made from hard glass capillaries and are assembled into a mouth-operated system made up of a mouthpiece, rubber tube (approximately 60 cm), and a pipet holder (components available from Sigma-Aldrich, A5177). 

Using the egg transfer pipet assembly, aspirate a little PBS, then pick up all the embryos in a minimal volume of PBS and transfer to the cryoprotectant. Do not expel bubbles into the dish. Allow to settle to the bottom for a minute or two. Distribute the embryos in groups of ten around the periphery of the dish. 

Use a pipet puller to prepare glass transfer pipets so that they are 2-3 cm long with a 120-180 pm diameter, and flame polish the end. Fill the pipet with a small amount of light paraffin oil, then an air bubble, then M2, followed by another air bubble. Fill the pipet with 5-7 eggs, followed by another air bubble and a small amount of M2. 

Locate the infundibulum under a dissecting microscope, and create a hole in the bursa with fine forceps. Pick up the infundibulum and insert the transfer pipet into the ampulla. Using a mouth pipet, expel the two bubbles flanking the eggs into the uterus. 

Follow the preceding procedure for making general-transfer pipets, but aim for pipets with internal diameters of around 120-150pm to permit easy passage of a mouse or rat egg. 

Using the general transfer pipet, transfer the eggs to a fresh dish of M2 medium to wash away traces of the hyaluronidase. Repeat in another dish of M2. In each wash, try to leave behind the cumulus cells. 

Remove about 20 one-celled eggs from storage in M16 medium at 37°C using a general-transfer pipet. 

Fig. 8. The lip of an oviduct transfer pipet enlarged to show the arrangement of eggs, air bubbles, and media.

Prepare the eggs for transfer. Remove a maximum of 20 microinjected eggs from the microdrop culture, and wash them in M2 medium. Load the eggs into an oviduct transfer pipet (Fig. 8), as described in the preliminary procedure. 

Expel the contents of the oviduct transfer pipete into the ampulla, and monitor delivery of the eggs by the appearance of the bubbles. When three bubbles have appeared, one can be certain that the eggs have been deposited into the ampulla. 

Remove poor-quality eggs with a plastic transfer pipet as these will decrease the quality of the overall extract. If greater than 10 % of the eggs appear poor in quality, the entire batch should be thrown away (reeNote 5). 

Draw some fluid into the pipet before drawing up the tissue piece. Otherwise the tissue may hit the meniscus and disintegrate. Then transfer the piece into the egg as quickly as possible. Do this on low magnification, and try to track the piece with your eye to avoid losing it. 

Transfer polarizing tissue graft into host egg using either a glass or gilson pipet or a very small spatula. 

Using a wide-bore Pasteur pipet, transfer eggs into Beckman ultraclear tubes. For these volumes, we typically use 14 x 95-mm mbes (Beckman, 344060). If multiple tubes are used, try to transfer an equal volume of eggs per tube. Allow the eggs to settle and remove as much CSF-XB as possible. 

Carefully cut through the oviduct, and squeeze eggs out using fine forceps. Transfer eggs to a Petri dish nsing a Pasteur pipet. Only use eggs from the oviduct do not dissect from the ovary. 

Transfer the eggs into prechilled 50 mL centrifuge tubes using a 25 mL pipet see Note 7). After the eggs have setded, the excess buffer should be removed. The eggs should remain covered with buffer. 

Transfer healthy larvae into a pipetting reservoir see Note 5). Exchange the medium with HEPES-buffered egg water several times to eliminate the tricaine (r Note 6). 

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