This document describes the Cre-loxP system for creating conditional gene knockouts in mice. The system involves introducing the Cre recombinase enzyme and loxP sites, which are not naturally present in mice. LoxP sites are inserted on either side of the gene to be knocked out in one "floxed" mouse line. A separate "Cre mouse" line is created that expresses Cre recombinase in a tissue-specific manner. Crossing the two mouse lines results in Cre recombinase recombining the loxP sites and deleting the gene of interest, but only in the tissues where Cre is expressed, allowing conditional rather than complete knockout of the gene.

1. GENE KNOCKOUT USING THE CRE-LOXP SYSTEM
BY KALYANI RAJALINGHAM
To create a conditional knock-out mouse, one needs the following systems: the Cre-loxP, and
the Flp-FRT system. First, let us focus on the Cre-loxP system which requires a Cre
recombinase, and loxP sites. The Cre recombinase is an enzyme that is required for
recombination; recombination between two loxP sites can induce a deletion.
The loxP site (34bp) has a central
8bp region (spacer region), and two
recombinase binding elements
(RBE); the two RBE are 13bp
inverted repeats. When there are
two loxP sites in the same direction
(Figure 1), recombination between
the loxP sites will delete the gene
of interest. Depending on the
orientation of the two loxP, one can create a gene inversion, translocation, or deletion. For
instance, if two loxP sites are placed on different chromosomes, a translocation can take
place. Thus, the orientation, and location of the loxP sites are important determinants of
outcome.
Both the Cre recombinase, and the loxP sites are not present in the natural mouse, and as such
must be introduced artificially. To do so, one mouse with the Cre recombinase (the Cre
mouse), and another with the loxP sites (the floxed mouse) are created, and crossed to

2. generate the Cre LoxP mouse. The Cre mouse can be designed with a tissue-specific
promoter for conditional knock-out. Alternatively, the Cre mouse can possess a non-
functional variant of the Cre recombinase that is rendered functional only when an inducing
agent is supplied. The floxed mouse possesses loxP sites on either side of the gene of interest.
Further, phenotypically, there should be no observable difference between the floxed, and
wild-type mice.
In order to create the floxed mouse, one must
first design the target vector, and inject into ES
cells; the target vector would have a 5’, and 3’
end that is homologous to the genomic DNA, a
marker gene, and the target gene flanked by the
loxP site (Figure 2). ES cells can then be
screened for resistance to a drug for instance –
those containing the marker will survive.
Subsequently, one must remove the marker; the
latter can be done by adding Cre recombinase to
the cells. In theory, it can generate 3 different scenarios – a single loxP site, two loxP sites
flanking the marker gene or the target gene. Screening using PCR should identify those cells
with the floxed allele. Those ES cells containing the floxed alleles can then be injected into a
blastocyst, and implanted into a foster mother.
The Cre mouse can be created in a similar fashion. Addition of target vector - with a tissue
specific promoter, the Cre recombinase gene, and an antibiotic gene flanked by FRT sites as
well as a HSV-tk site – to the genomic DNA is followed by recombination, and selection of

3. those cells with an incorporated target vector segment. Selection is carried out by growing the
cells on an antibiotic. Removal of the marker gene is achieved by adding the FLP
recombinase enzyme which deletes the marker gene. The Cre+ ES cells can then be injected
into a blastocyst, and implanted into a surrogate mother. Depending on the orientation of the
two loxP, one can create a gene inversion, translocation, or deletion. For instance, if two loxP
sites are placed on different chromosomes, a translocation can take place. Thus, the
orientation, and location of the loxP sites are important determinants of outcome.
Usually, the Cre-loxP system is used when simple knock-outs would result in the death of the
cluster of cells; in other words, when you need to examine a gene that is required for
embryonic development, but that we also wish to study the gene, we resort to the Cre-loxP
system – a conditional tissue-specific knock-out system.