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

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

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.