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RESEARCH DIVISION Laboratory Manual

 


 

Collection of Morulae and Earlier

Before you start, have ready the following:

- Sterilize 2 pairs of curved forceps, 1 pair of iris scissors and 2 pairs of fine watchmaker forceps.
- 6ml syringe filled with DMEM with HEPES, with an 18 gauge needle attached. To the needle attach a 20cm length of clear vinyl tube and into the end of this tubing insert a sharp flusher.

At 2.5 days p.c. (post coitus) the morulae are present in the oviducts. For this reason, it is only necessary to remove the oviducts from the mouse.

Kill the 2.5 day pregnant mouse and lay on its back on benchcloth or absorbent paper. Swab belly with ethanol (70%) and nick the skin with a pair of scissors. Pull the skin back over the head and towards with tail. Cut and pull back the body wall to expose the contents of the abdomen. Push aside the guts to expose the reproductive tract. Using a pair of curved forceps, grasp the uterus just below the oviduct and carefully separate the oviduct from the ovary using the iris scissors. Cut through the uterus just below the uterotubal junction and place the oviduct in a petri dish.

Transfer the dish to the stage of a stereo dissecting microscope. Using the watchmaker forceps, manipulate the oviduct so that you have the end of the oviduct (close as possible to the uterotubal junction) between the forceps and insert the flusher so that it points away from the uterotubal junction. Be careful not to pierce through both sides of the oviduct tube. Keeping the flusher inserted into the oviduct, pick up the syringe in the other hand and give a couple of short, sharp squeezes on the plunger. This should flush all morulae from the oviduct. Sometimes, if no morulae are flushed through, it is hard to tell whether the oviduct has been correctly flushed. If there is much debris floating around, then the oviduct has been properly flushed. The morulae are then collected using a mouth pipette and deposited into a petri dish in microdrops of M16 medium. These drops are covered with fluid 200 (Dow Corning, viscosity 50CS) and placed in a 37C incubator buffered with 5% CO2.

Tubing for Flusher

- Single lumen
- Clear vinyl tube, medical grade
- Cat. No. S.V. 95
- ID = 1.2mm
- OD = 1.7mm
- OR Cat. No. 111074 ID=1mm, OD=2mm

Made by

Dural Plastics and Engineering
PO Box 21
Auburn 2144

Glass for Flusher/Transfer Pipette

Made by Clark Electromedical Instruments
Type: GC120-15 (350 pieces per tube)
Distributed by SDR Clinical Technology (02) 958-2688
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