Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 237
8. Spin down gently (90g; 5 min) and remove supernatant.
9. Add dropwise 2 to 3 mL of chilled fixation solution (3:1 methanol/acetic acid,
freshly prepared) onto the pellet, resuspend by flicking, and incubate 5 min at RT.
10. Spin down gently for 5 min at 90g; and remove supernatant.
11. Repeat fixation procedure once or twice.
12. Finally, resuspend pellet in 0.5–1 mL of fixation solution, drop the solution onto
clean slides from a distance of 15–30 cm, and air-dry slides.
13. Check under the microscope if the broken nuclei are sufficiently separated from
each other to ensure that every chromosome can be assigned to its metaphase spread.
14. Incubate slides for 1 min in Giemsa staining solution (0.4% modified Giemsa
stain, Sigma).
15. Wash twice with distilled water, air-dry, and mount the slides.
16. Count the number of chromosomes in metaphase spreads of the nuclei. Because
direct counting under the microscope is tedious and error prone, it is recommended
that pictures be taken on which the counted chromosomes can be
marked.
3.9. Preparation of HM-1 Cells for Blastocyst Injections
A subconfluent 25-cm2 flask of cells is sufficient. Ideally, thaw cells 4 d
before injection. Approximately 24 h before microinjection, they should be near
confluent.
1. Trypsinize and replate 70–100% of cells.
2. Change medium early in the morning of the injection. Start trypsinization only
when everything at the microinjection facility is ready to go. Keep in mind that
cells become fragile after 1.5–2 h.
3. Trypsinization: aspirate medium, add 2 mL of trypsin, mix gently, and remove.
Add 3 mL of trypsin and incubate 5–10 min at 37°C to receive single-cell suspension
(check under microscope and continue incubation if necessary).
4. Add 5 mL of medium and pipet up and down gently.
5. Spin 5 min at 250g, aspirate medium, and resuspend cells in 5 mL of injection
medium (GMEM, 10% FCS, 20 mM HEPES, 1X Pen/Strep).
6. Before adding cells to microinjection droplet, add 1 µl of ultrapure DNase I
(20–40 U/µL). Keep cells on ice during injection.
3.10. Production of Chimeric Mice
The setup for the injection of blastocysts or the production of aggregation
chimeras has been extensively described previously (127).
3.10.1. Preparation of Hormones for Superovulating Female Mice
Prepare stock solution in sterile 0.9% NaCl. Keep solution frozen in convenient
aliquots at –20°C until use (hormone stocks are stable in the freezer for
more than 1 yr). Thaw before use and do not refreeze. If aliquots are not used
in full, discard them after injection.