Target Discovery and Validation Reviews and Protocols

226 Vijayaraj, Söhl, and Magin
gene and the alteration is shorter than between the alteration and the end of the
homology arm. One potential drawback of this strategy is that the expression of
the target gene can be severely altered by placing the marker gene in regulatory
introns (85,111,112).
An alternative strategy follows the so-called double replacement procedure
(113–115) (Fig. 4C). In the first round of gene targeting, an HPRT-minigene
replaces the (part of the) gene to be modified. After the first round of correct
targeting, the minigene is replaced by a modified version of the original gene or
by another sequence. In the second step, selection with 6-TG allows cell survival
only upon loss of the HPRT-minigene. If HPRT-deficient ES cells are not
available, a combination of two selectable marker genes for positive and negative
selection has been described previously (113,115). In principle, this procedure
allows the introduction of many point mutations by using the same
second-round gene-targeting vector. In contrast to Cre-Lox or FRT/Flp procedures,
no foreign DNA remains in the target locus (Fig. 4D).
With the use of the site-specific recombinases, conditional cell type and temporal
gene alterations have become feasible (116–120). The original vector
design used a Lox P-flanked marker gene and a third Lox P site placed in an
intron such that the Lox P sites flank an exon to be deleted (117). After Crerecombinase
activity, three types of excisions can occur (Fig. 4D), of which complete
and type II excisions are the desired excisions and are selectable by the loss
of the marker gene. The type II excision provides a conditional allele that can be
deleted either in ES cells or in vivo, after mating with a strain of mice expressing
the Cre-gene under the control of an appropriate promoter. Because type II excisions
can be difficult to obtain, an improved strategy relies on the combined use
of Lox P and FRT sites. FRT sites are used to flank the selectable marker gene
and allow its removal by the Flpe gene (121), either in ES cells or in vivo. Two
Lox P sites are placed at intronic or noncoding exon positions to delete (parts of)
the gene of interest (Fig. 4E). Conditional alleles created in this way can be controlled
in a temporal manner by using an inducible Cre-recombinase. At present,
tamoxifen, RU 486, and tetracycline-controlled Cre-recombinases, in combination
with various promoters, are available. Currently, there are two limitations of
these inducible transgenes, namely, leakiness at the uninduced stage and the time
to remove the target gene product after Cre expression. It can take several days
for this process, especially for IF proteins with their long half-lives (122). The use
Fig. 4. (Continued) occur after Cre activity, depending on the cell cycle phase in which
Cre is active and the chromosomal integration of the two targeting vectors (123). Below,
after Cre activity, the marker genes and the genomic sequences between the two lox P
sites are deleted and surviving, correctly targeted ES cell clones are resistant against HAT
and sensitive against G418 and puromycin.