Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 149

Fig. 1. Example of a gene-targeting strategy. The wild-type allele is substituted by
a targeting construct that silences the gene. The null mutation is generated by insertion
of the neomycin resistance gene into the sequences of exon1 . R indicates the site rec-
ognized by a single restriction enzyme. Probe indicates the genomic fragment used for
identification of homologous recombinants. As shown in the framed panel, in contrast
to wild-type ES clones that show only the high molecular weight band (+/+), heterozy-
gous cells show also the targeted allele-specific shorter band.
The probe must generate a diagnostic signal that distinguishes the targeted
allele from the wild-type counterpart. This result can be obtained by finding a
restriction enzyme that cuts outside the construct and generates a fragment that
encompasses the probe. In the best situation this same enzyme cuts the targeted
allele inside the resistance cassette and generates a fragment that is shorter
than the wild-type ( Fig. 1 ). Generation of shorter segments is recommended
because the identification of a recombinant clone will not be confused by the
presence of partially digested DNA.
If the search for a probe with the above features fails, it will be necessary to
change the strategy outlined above and find alternatives such as extending the
restriction map to new enzymes and considering other exons, deletions or
resistance cassettes. Because cloning steps involved in construct preparation
are often complex and time consuming, it is wise to start to build the construct
only if a good probe has been found.
3.2. Mutations Using the Selection Cassette
In classical constructs, null alleles are made by inserting the neomycin resis-
tance cassette in the middle of an exon. The choice of an exon near the 5' end of
the gene enhances the chances of generating a nonfunctional product. Alterna-