Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 151

Fig. 2. Example of conditional gene targeting using the Cre/loxP technology. The
wild-type allele is replaced by a targeting vector in which the resistance cassette and
exon 1 are flanked by three loxP sites (filled triangles) showing the same orientation.
R indicates a restriction enzyme site. Probe 1 serves to identify homologous recombi-
nant clones after digestion with that enzyme. Transient expression of Cre allows gen-
eration of a loxP containing (active) and deleted (inactive) allele. Probe 2 identifies
different Cre-mediated recombination events in R digested genomic DNA. Excision
of the DNA segment comprised between the outer loxP sites leads to a null allele
(type I deletion). Recombination involving loxP sites flanking the resistance cassette
generate a conditional null allele (type II deletion). A third possible deletion involving
exon1 only is not shown. Mice homozygous for the floxed conditional null allele must
be normal. Breeding with Cre expressing transgenic mice produces an offspring that
should show the induction of null alleles.
tive/negative selection cassette can be used to ease the identification of deleted
clones. Targeted clones are isolated in the presence of G418 ; successively,
clones that lost the cassette are selected using gancyclovir. To identify
the cells where Cre exerted its function, it is essential to find a restriction
digest and a probe that can distinguish between the deleted and the original
targeted alleles ( Fig. 2 ).
3.3. Manipulation of ES Cells
ES cells to be used for knock-out experiments must be kept in an undifferen-
tiated state. ES cells are small and round, with a large nucleus and few cyto-