Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 150

tively, the resistance cassette can be introduced in place of some genomic DNA.
It has been reported that deletions of up to 10 kb are compatible with homolo-
gous recombination in ES cells (29) . The resistance cassette should be always
inserted in an area that is not subjected by alternative splicing. It must be also
kept in mind that the insertion of the cassette can cause aberrant splicing events
that either generate out-of-frame mutations (22) or the skipping of the mutated
exon. To avoid this latter case, a good strategy implies the insertion of the
cassette in exon 1 .
Several variations of the neomycin resistance cassette have been shown to
function in targeting constructs. Consistent successful results have been
obtained using the strongly active PGK (phsphoglyceratekinase) promoter
driving the expression of the wild-type bacterial neomycin-phosphotransferase
gene. Although weaker cassettes have been used, high-performance selectable
genes are preferable: they allow the screening of more clones per single
transfection experiment and are less susceptible to downregulation by the tar-
geted locus.
3.2.3. Mutations Using loxP Sites
The use of the Cre/loxP system greatly enhances the ability to generate tissue
specific knock-outs. This technique allows the creation of mutations that can
be induced under controlled conditions (9-11) , the generation of homozygous
mutant ES cells ( see Section 3.8. ), point mutations (30) , or large deletions
(31) . These sophisticated gene-targeting experiments require the insertion of
loxP sites in significant regions of the locus of interest. The loxP site is a
34-bp-long inverted repeat (5'-ATAACTTCGTATAGCATACATTATACGA
AGTTAT-3') that is recognized by the Cre recombinase (8) . In the presence of
two loxP sites oriented in the same direction, this enzyme deletes the genomic
DNA between the inverted repeats reforming a single functional loxP site. If
loxP sites are in opposite orientations, the Cre recombinase reverts the inter-
vening sequence. Constructs for conditional knock-outs should usually contain
at least two loxP sites that flank a DNA segment, which once deleted or flipped,
leads to gene inactivation. For this purpose, loxP sites can be placed in
noncoding regions so that they flank one or more exons ( Fig. 2 ). In classic
constructs three loxP sites are used: two are contained in a floxed selection
cassette and a third is placed within one homology arm. This latter site must
not be too far from the other two. It has been reported that integration by
homologous recombination of this loxP site strongly decreases with the dis-
tance from the nonhomology region. The presence of floxed selection cassette
allows to eliminate heterologous DNA from the targeted locus, leaving a pre-
sumptive functional allele. Cre mediated excision of the resistance cassette
( see Section 3.6.5. ) is a relatively efficient process; nonetheless, a neo-tk posi-