Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 165

1. Thaw heterozygous ES cells carrying a neomycin resistance cassette on one allele.

Grow and expand on feeder to obtain at least 105 cells.

2. Plate ES cells on 10 9-cm tissue-culture dishes with 10 4 cells each.

3. The next day, change medium with ES medium additioned with 4 mg/mL G418.

4. Change medium every day. Check for dying cells. If cells do not start to die in

4-5 d, it is likely that the technique will not succeed.

5. Wait for 6-7 d to start to see colonies. Treat resistant clones as described in

Subheading 3.6.3.

6. Analyze the genotype as described in Subheading 3.6.4.

3.7.1. Analysis of ES Cell Contribution in Chimeric Mice
Genetic differences between ES and host embryo derived cells can be
exploited to measure contribution of the injected cells in a chimeric tissue.
Allelic variants of glucose phosphate isomerase (GPI) enzymes are often
utilized for these measurements because they show distinct electrophoretic
mobility on a cellulose acetate plate. The C57B6 derived GPI-B presents a
higher electrophoretic mobility compared to the 129 derived GPI-A ( see Note 17 ).
1. Dissect a small piece of tissue (10 mg or less) from the chimeric mouse.

2. Lyse the sample in 100-200 L of extraction buffer using a small pestle. Lysates

can be stored indefinitely at -20

µ

° C.

3. Mark the upper side of the plastic back of the plate. Soak the Titan III

plate in Supre Heme buffer avoiding the formation of air bubbles inside the
cellulose acetate.

4. Dilute the sample to the desired concentration and load 8 µ L on the Super Z

well plate.

5. Recover the Titan III plate and blot it dry between two paper towels. Fix the plate

on the loading device with the cellulose on the top and so that samples will be
loaded near the marked side.

6. Collect some sample by pressing the applicator inside the wells. Blot the applica-

tor on tissue paper. Reload in the same way the applicator and finally press it
against the cellulose acetate plate.

7. Fill buffer tanks of an electrophoresis device with Supre Heme buffer. Place a

piece of filter paper in both buffer tanks so that they do not touch each other.

8. Overlay the plate onto the two pieces of filter paper so that the cellulose side

faces the bottom of the chamber. In this way, the plate is electrically connected to
the buffer tanks. Take care to place the marked side near the anode. Stabilize the
extremities of the gel with 5-8 glass slides. Run (from anode to cathode) at 200 V,
4 ° C for 3 h.

9. Place the Titan III slab onto a glass plate. Melt the agarose and cool 10 mL at

55 L of each developing reaction component, mix well and pour it

over the cellulose acetate plate. Leave the reaction in the dark for 2 to 15 min,
depending on the concentration of samples.

° C. Add 200 µ

10. Place the plate in stop solution as soon as the bands reach the desired intensity.

Photograph immediately.