1The Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA, 2ParaTechs™ Corp., University of Kentucky, Lexington, KY, USA, and 3Department of Microbiology, Immunology, & Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, USA
Peer-reviewed,author-submitted protocol Published in November 2009 (p.52) DOI: 10.2144/000113259
Instructions for use
Prior to nonsurgical embryo transfer
For the production of zygotes and pseudopregnant females to serve as recipients, standard transgenic methodologies are used. Matings are set up with male and female mice as in standard transgenic procedures. Female donors can be superovulated if desired. Embryos should be 3.5 days post coital (dpc) on the day of nonsurgical embryo transfer (NSET); recipient females should be 2.5 dpc on the day of NSET transfer. Embryos are incubated in KSOM media (Cat. no. MR-106-D; Millipore, Billerica, MA, USA) or similar.
Protocol
1. Place a 15-L drop of KSOM onto the lid of a 100-mm Petri dish [BD Falcon (Cat. no. 351029; Franklin Lakes, NJ, USA) or similar]
2. Load 20 blastocysts into the KSOM drop using a standard embryo handling pipet. (Note: optimal number of embryos to transfer may vary depending on mouse strain and the types of manipulations embryos have received.)
3. Place the NSET device (Figure 1) onto a P-2 Pipetman that has been set to 1.8 L.
4. Press Pipetman plunger to first stop, lower tip into media, and slowly pull embryos into the tip of the NSET device. Visualize embryos using dissecting microscope.
5. Carefully set the Pipetman to 2.0 L to create a small air bubble at NSET tip (this helps prevent embryos from wicking out during NSET insertion into mouse). Gently lay Pipetman with loaded embryos on its side near mouse cage.
6. Place the unanesthetized recipient female on the top of a cage, allowing the mouse to “grab” the surface with its forefeet. The metal cage top works well for this. Grasp the midpoint of the tail using thumb and forefinger, and angle the tail upward while lightly pressing the base of the tail with the opposite edge of the hand.
7. Gently place a smaller piece of tubing into the mouse’s vagina, then remove quickly. This will help open the vagina.
8. Place speculum into vagina. Using an adjustable light to shine into the speculum, locate the cervix (1). We use the Nikon MKII fiberoptic lamp for this purpose.
9. While holding the female mouse with one hand as described in Step 6, carefully pick up the Pipetman and insert the NSET tip into the speculum, through the cervix and into the uterus (Figure 2). Once the NSET hub contacts speculum, expel embryos by pressing plunger to first stop. Inserting the NSET device is more problematic with smaller recipient mice, due to the smaller size of the cervix. It is important to place the speculum properly and visualize the cervix using the lamp.
10. Immediately remove the NSET device and speculum. Return mouse to cage.
References
1. Popesko, P., V. Rajtová, and J. Horák. 1992. A Colour Atlas of the Anatomy of Small Laboratory Animals, Volume 2: Rat, Mouse, Hamster, p. 160. Wolf Publishing, London.
Address correspondence to Brett Spear, Room 210, Combs Building, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY, 40536-0298, USA. email: [email protected]
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