THE DERIVATION AND MANIPULATION OF EXPERIMENTAL ANIMALS IN BIOMEDICAL SCIENCES - A Guide to Methods in the Biomedical Sciences - page 111

sequence, such as that containing the neo gene, is flanked by LoxP
sites (flanked LoxP sites are “floxed”), addition of the Cre recombinase
results in its deletion. To remove the intervening sequences flanked by
loxP sites in ES cells, a vector containing the Cre recombinase can
be introduced into the ES cells by electroporation. Figure 11 shows the
removal of the selectable neo gene using this technology.
Conditional mutants using floxed genes
The use of the Cre-lox system has now evolved well beyond its initial
use in removing the selectable marker from targeting vectors in ES cells.
Today, this system is used to delete genes in an inducible or tissue spe-
cific fashion to conditionally delete genes. This technology is especially
useful in the studies of genes that affect development, since knockouts
of these genes often lead to embryonic lethality. To generate conditional
mutants, two different approaches are used.
In the first approach, two different transgenic mouse lines are inde-
pendently generated, one carrying the Cre recombinase under the con-
trol of a tissue specific promoter, and a second carrying the floxed gene.
The two strains of mice are mated. The floxed gene is deleted in the
progeny mice, but only in the tissues in which the Cre recombinase is
expressed. Currently, there are a number of Cre transgenic mouse lines
that express the recombinase under the control of different promoters
that have very selective tissue specificity.
A second approach utilizes mice in which the Cre recombinase is
under the expression of an inducible promoter. In these systems, floxed
genes are deleted only when the inducible promoter is activated, there-
fore activating the expression of the recombinase. This is especially
useful if the investigator wishes to delete a gene only after it has been
expressed, as a means to determine the role that particular gene product
plays after the development of a tissue. Inducible promoters that have
been used for these purposes include response elements that are acti-
vated by antibiotics, such as tetracycline, or hormone analogues, such
as tamoxifen (the estrogen antagonist that is used for the treatment and
prevention of breast cancer).
The FLP-FRT system
The FLP-FRT system is very similar to the Cre-lox system and is
becoming increasingly popular in the generation of knockout mice, ei-
ther alone or in combination with the Cre-lox system. FLP is a flippase
recombinase isolated for the yeast Saccharomyces cerevisiae, and
FRT is the f lippase r ecombination t arget sequence. When genes are