Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 239
power to overcome an embryonic lethal phenotype that is caused by an
extraembryonic defect, if tetraploid wild-type embryos are aggregated with
diploid knockout ES cells (55). The principle of this experiment is outlined in
Hesse et al., 2005 (32). If a homozygous ES cell line of the knockout of interest
is not available, knockout embryos of the eight-cell stage can be used for
aggregation. The efficiency is significantly lower, compared with aggregation
with ES cells, because the embryos can still contribute to the extra embryonic
compartments.
3.11.2. Recovery of Two- and Eight-Cell Embryos
Two-cell embryos are flushed out on day 1.5 p.c., whereas eight-cell
embryos are flushed out 1 d later at day 2.5 p.c. To perform these experiments,
the following additional materials are needed: dissecting microscope, flushing
needle (the sharp tip of no. 30-guage 1/2 needle is cut off and then rounded
using sharpening stone), 1-mL syringe, dissecting instruments (fine-pointed
scissors, fine forceps); 5 forceps (Dumont), mouth pipet (aspirator mouth piece,
latex tubing, blue tip, micropipette), sterile tissue culture dishes (35 × 10 mm),
and M2 and KSOM-AA media.
1. Superovulate an appropriate number of CD-1 females (see Subheading 3.10.1.),
mate with CD-1 males, and check for copulation plugs.
2. On day 1.5 or 2.5 post coitus, sacrifice the plugged females by cervical dislocation
to obtain two- or 8-cell embryos, respectively.
3. Dissect the oviducts by cutting the upper part of the uterus and underneath the
ovary.
4. Place oviducts in a 35-mm tissue culture dish filled with M2 medium at RT.
5. Transfer an oviduct into a tissue culture dish with M2 medium under a dissecting
microscope.
6. Connect the flushing needle to a 1-mL syringe and fill it with M2 medium.
7. Grab the infundibulum with a fine forceps and insert flushing needle.
8. After successful insertion, hold the needle with the forceps.
9. Flush out oviduct with 100–150 µL of M2 medium by applying constant pressure.
10. Repeat all steps for each oviduct dissected.
11. Spin the tissue culture dish containing the embryos manually with fast circular
movements. This motion brings the embryos to the middle of the dish.
12. Collect all embryos with the mouth pipet and rinse them trough 3 drops of
M2 medium and 3 drops of equilibrated KSOM-AA medium.
13. Store the embryos in KSOM-AA drop under mineral oil in an incubator (37°C;
5% CO 2 ).
3.11.3. Generation of Tetraploid Embryos
Fusion of the blastomeres of two-cell embryos occurs when a square pulse
is applied perpendicular to the plane of contact of the two cells. Adjustable