Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 235
16. Grow to near confluence.
17. Trypsinize and plate onto two 25-cm 2 flasks adding two-thirds on one flask and
one-third to the other flask. When confluent, use the two-thirds flask for freezing
cells (two to three vials) and the other flask for preparation of genomic DNA.
18. Maintain selection until targeted ES cells are expanded and characterized by
Southern blotting.
19. To remove cells from HAT selection, replace HAT with HT for at least 2 d before
growing cells in normal medium. Cells grow slightly faster in the absence of selection.
For HPRT negative selection, the final concentration of 6-TG is 5 µg/mL.
20. For correctly targeted ES cells, thaw an aliquot on 25-cm 2 flask; grow until confluent,
and passage on 1X 75-cm 2 flask. Prepare six to eight freezing aliquots for
blastocyst injections.
3.5. Double Replacement by Using HPRT-Minigenes
(Second Step of Gene Targeting)
1. Keep passage number between first and second gene-targeting step as low as possible
to minimize the accumulation of 6-TG–sensitive cells before electroporation.
2. Electroporate approx 20 million cells as in Subheading 3.4. and plate onto six
75-cm2 flasks.
3. Apply 6-TG selection at day 6. Most likely, it will be necessary to split cells before
selection. Split in such a way that you end up with six near confluent flasks at day 6.
Then, trypsinize cells.
4. From each flask, plate 6 × 1–1.5 million cells onto 10-cm dishes in medium containing
6-TG.
5. Discard remaining cells.
6. Change medium after 2 and 4 d, respectively.
7. Colonies grow approx on day 10 (there will only be one to five colonies per dish).
Process resistant colonies as described in Subheading 3.4.
3.6. Isolation of ES Cell DNA for PCR-Based Genotyping
With good vector design and the use of isogenic DNA for vector construction,
high gene-targeting frequencies can be expected. For a range of different
genes, we noted frequencies of 1:5 to1:35 PCR-positive colonies. Therefore, it
should be sufficient to analyze between 100 and 200 colonies per experiment.
The procedure detailed here is based on using approximately half of a colony
for DNA isolation and the other half for further growth. In this way, positive
colonies can be scored the next day. For analysis, small, slower growing ES cell
colonies with a good morphology should be used.
1. Label colony position with a marker pen and isolate them with a yellow Gilson tip
under the clean bench.
2. Coat 24-well plate with gelatin and remove by aspiration after 10–20 min.
3. Pick colonies along with approx 150 µL of medium by using a P 200 Gilson pipet.