Target Discovery and Validation Reviews and Protocols

172 Houdebine
2.2.3. Knock-In Into Hypoxanthine–Guanosine Phosphoribosyl
Transferase (HPRT) Locus
Targeted DNA integration is mainly used to knock out genes. It may be implemented
as well to introduce an active gene in a given site of a genome. This operation
known as gene knock-in allows reproducible expression of transgenes
always subjected to the same position effect. The HPRT locus is one the sites in
which foreign genes are successfully targeted in mice (Fig. 3). This gene is
expressed in all cell types. Interestingly, the HPRT locus allows not only a satisfactory
expression of foreign genes designed to be expressed in all cell types but
also proves to direct the expression in animals of a variety of gene constructs containing
cell specific promoters (46). A company is extensively using this technique
to express successfully foreign genes for academic or industrial laboratories.
2.2.4. Use of Cre-Lox P and Flp-FRT Systems
Two systems allow the integration of foreign genes into selected sites of
genomes. One system known as Cre-LoxP is of bacterial origin and the other
system, Flp-FRT, comes from yeast. In these two systems, the short LoxP and
FRT DNA sequences are first introduced into a genome, either randomly or in
a targeted manner. Foreign genes containing either LoxP or FRT sequences can
integrate specifically in the homologous sites previously introduced into genomes
under the action of the specific recombinases Cre and Flp, respectively. The
LoxP and FRT sites in the genome may be retained after the demonstration that
they favor transgene expression. Alternatively, the two LoxP and FRT sites may
have been introduced previously in vectors containing elements able to favor
transgene expression such as insulators.
These two systems require the use of two different sites added on both sides
of the foreign gene. This technique known as recombinase-mediated cassette
exchange allows integration of the foreign gene into the genome but not its
deletion, which spontaneously occurs with a single site (Fig. 3; [47,48]).
Notably, the Cre-LoxP and the Flp-FRT systems allow the conditional deletion
of a gene. For this purpose, two LoxP or FRT sites must be introduced in
the periphery of the gene to be deleted in the genome. This approach implies
two homologous recombinations. The expression of the Cre or Flp recombinases
in given tissues induces the specific knockout of the targeted gene. This
knockout can be achieved by crossing mice harboring the LoxP or FRT sites
with other mice expressing the recombinase genes in the chosen tissues.
Alternatively, adenoviral vectors harboring the recombinase genes may be
injected into the tissues in which gene knockout is desired.
The Cre-LoxP and the Flp-FRT systems allow the deletion of long DNA
fragments from a genome and chromosome crossover. In the first case, LoxP or