Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 163

5. Collect the DNA containing supernatant as in step 4 and add it to a fresh tube

containing 0.5 mL of isopropyl alcohol. Invert the tube a few times and pellet
the white DNA precipitate to the bottom by a short spin. Carefully eliminate the
alcohol and air-dry the DNA for a couple of minutes.

6. Add to the DNA pellet 100 µ L of sterile DDW and resuspend it overnight at

56 ° C. Store the DNA solution at 4 ° C.

7. Analyze the DNA samples as described in Subheading 3.3.4., steps 6-13 . Using

the probe defined for the screening of targeted ES clones, homozygous and het-
erozygous mice show one or two bands, respectively.

3.6.2. DNA Preparation for PCR Analysis
1. Design three oligonucleotides amplifying two short (<500 bp), but distinct diag-

nostic fragments each characteristic of a single allele. One oligonucleotide should
be common and the other two should anneal to wild-type and mutant DNA
sequences, respectively ( Fig. 4 ). Best results are obtained with 20-mers having at
least a 50% GC content.

2. Label mice as in Subheading 3.6.1., step 1 . Collect no longer than 2-3 mm of

the tip of the tail in a 1.5-mL tube containing 50 µ L of lysis buffer.

3. C shaking in the Thermomixer for 2-3 h. Smash rem-

nants of tail using a fresh yellow tip for each tube. Inactivate proteinase K by a
6 min incubation at 95

Incubate samples at 60 °

° C. Spin at maximum speed for 5 min. Dilute 5 µ L of the

supernatant into 10 µ L of sterile DDW.

4. Set up a PCR reaction following standard procedures (27) using 5 µ L of the

diluted sample in a final volume of 50 µ L ( see Note 15 ).

5. Amplify using the following cycle profile.

• 10 cycles 95 ° C, 30 s

65 ° C-1 ° C each cycle, 30 s

72 ° C, 30 s

• 25 cycles 94 ° C, 30 s

55 ° C, 30 s

72 ° C, 30 s

6. Check the reaction on 2% agarose gel. Amplification of one band with both com-

bination of oligonucleotides indicates heterozygosity. Samples showing a band
with either one of the two couples of oligos are to be considered wild-type or
mutant homozygous depending on the length of the diagnostic fragment detected
and on the presence of the wild-type or mutant allele specific oligonucleotide in
the productive reaction.

3.7. Generation and Analysis of Double KO ES Cells
Knocking-out genes essential for embryonic development leads to lethality
before birth and in many cases precludes the analysis of gene function in adult
tissues. To overcome this limitation, it is necessary to generate Cre/lox condi-
tional mutagenesis. A simpler alternative is to isolate ES cells homozygous
for the mutation, which can be used to generate chimeras. The analysis of the