Target Discovery and Validation Reviews and Protocols

234 Vijayaraj, Söhl, and Magin
b. Irradiate cell suspension at 15 Gy for 7 to 8 min.
c. Replate irradiated cells on 10–15 75-cm 2 gelatinized flasks overnight.
Trypsinize and freeze in aliquots or use in experiment.
For use in ES cell growth, plate feeders on gelatin-coated dishes 1 to 2 h
before plating ES cells. A density of 50,000 cells/cm2 is required. Feeder layers
last approx 1 wk after treatment.
3.4. Electroporation of ES Cells (First Step of Gene Targeting)
The conditions recommended will work with a Bio-Rad gene pulser and
cuvettes of 0.4-cm electrode gap. Setting: 800 V, 3 microfarads (standard). The
conditions will work with any ES line and are relatively insensitive to changes
in cell number and amount of DNA. They do not yield the highest possible
number of transformed cells, but they are gentle to cells. Other settings that
yield higher efficiencies are 250 V, 500 microfarads (optional). Under these
conditions, use 25 µg of DNA and 106 cells per electroporation. Changes in
DNA amounts and cell number alter efficiency and cell viability. Optimal
results are currently achieved if the DNA is purified with Nucleobond or QIA-
GEN columns. EndoFree purified DNA is not necessary, but it may improve
transfection efficiency.
1. For a standard targeting experiment, trypsinize approx 20–30 million cells.
2. Determine cell number in a counting chamber.
3. Resuspend ES cells in 0.8 mL of 1X HBS buffer.
4. Add cells to 200 µg of linearized DNA in 100 µL of sterile TE in Eppendorf tube
(phenol/chloroform-extracted, ethanol-precipitated, remove 70% ethanol in clean
bench).
5. Mix by pipeting up and down using a 1-mL cell culture pipet.
6. Transfer into electroporation cuvet.
7. Apply one pulse (standard or optional) and allow to stand for 10 min at RT.
8. Add cells to appropriate amount of medium and plate approx 1 to 2 × 10 6 cells/
10-cm dish in 10 mL of medium.
9. Start positive selection the next day (refer to additional reagents in Subheading 2.).
Leave one plate without selection to check cell recovery.
10. After 3–5 d, change medium again (electroporation is day 1). Colonies will grow
after 8–10 d.
11. Isolate colonies as described in ES cell colony isolation protocol (Subheading
3.6.), use half for PCR and leave the remaining cells in 24-well plate with approx
1 mL of medium.
12. After a few days, multiple colonies should have grown.
13. Aspirate off medium, wash with 0.2 mL of trypsin, and aspirate off again. Add 2
to 3 drops of trypsin and incubate for 3–5 min at 37°C.
14. Add 0.5 mL of medium and break up cell clumps by using a blue-tip Gilson pipet.
15. Transfer cells to a six-well plate and add 5 mL of medium.