CONCLUSIONES

One o f the most widespread uses of the site-specific recombination system is in gene targeting strategies. Using homologous recombination specific, genes are targeted in embryonic stem (ES) cells. These cells can later be used for the generation of mice lacking the gene targeted (gene knockout) (Muller 1999).

In addition to the alteration introduced in the genome, it is essential for a selectable marker gene to be co-introduced. The product of the marker gene is used for the identification o f the targeted ES cells. However, this marker gene can and often does interfere with gene expression of the targeted gene, as well as with neighbouring genes (Fiering, Epner et al. 1995; Tiering, Bender et al. 1999). It has been reported for instance, that introduction o f a neomycin resistance gene (neo), as a selection marker in a knockout strategy, reduced the expression of the targeted gene at variable levels (Nagy, Moens et al. 1998). This usually means that hypomorphic alleles o f the targeted gene are created, which do not abolish its expression completely, as required. Cre recombinase can help to overcome this problem. All that is required is the incorporation of loxP sites flaking the selectable marker in the targeting vector. After incorporation of the vector in ES cells and selection of the targeted clones, transient expression of Cre protein removes the marker leaving a loxP site behind (Figure 1.9 A). This loxP site does not seem to have any effect in the expression of the gene o f interest or its neighbours.

Simultaneously, this method permits the use of the hypomorphic alleles, generated by the presence of the marker, in a constructive way. Using different breeding schemes and expression of the Cre recombinase, mice bearing a combination of such an hypomorphic allele with null or wild type allele can give rise to lines with variable levels of the targeted expression. The importance of such an analysis is exemplified in the case of N-myc gene (Nagy, Moens et al. 1998). Null mutations of this gene are embryonic lethal. By employing the above strategy, Nagy and co­ workers managed to generate mice with reduced levels of expression of N-myc. These mice were viable and their analysis revealed a role of N-myc in the development of a variety of tissues, such as the lung, heart and skeleton. Furthermore, in mice carrying

the hypomorphic alleles Cre expression could excise the neo marker, reconstituting normal levels of gene expression. This conversion of the mutant phenotype to the wild type holds further promise for use in a Cre mediated gene repair strategy, where a

gene is mutated and corrected in situ. Such an approach would expand the potentials

for the analysis of complex multiple gene functions.

Still, the most promising use of the Cre/loxP system at present lies in the generation o f conditional knockout mice (Rajewsky, Gu et al. 1996; Rossant and McMahon 1999). These are mice that lack a certain gene, only in a specific tissue or cell line and at certain time during development or adult life. The strategy relies on the concept of flanking the gene o f interest or essential part of it with loxP sites (Figure 1.9 B). The integration of loxP sites should not influence gene expression and mice carrying the floxed (flanked by loxP sites) allele should appear to be normal. Expression of the Cre recombinase in a site and time restricted way results in the inactivation of the gene at a site and temporal controlled way.

In a refined development of this approach, both the gene and the selection marker gene are flanked by loxP sites. Cre expression and selection o f the appropriate recombination event gives rise to the proper mutation. Although this idea appears to be simple, there are many complications especially, in regard to achieving the appropriate Cre recombination events. Recently, along with the advances in the FLP recombinase (Buchholz, Angrand et al. 1998; Buchholz, Ringrose et al. 1996), the favoured strategy is to use a combination of FLP and Cre recombinase. More precisely, FLP recombinase can be used for the removal of the selection marker and Cre recombinase targeted to a specific tissue for the generation o f the conditional mutation (Rodriguez, Bucholz et al. 2000). Such a complex strategy was applied successfully for the analysis of the FGF8 (Meyers, Lewandoski et al. 1998). A transgenic line was created (allogenic) carrying an hypomorphic allele, which could give rise to the null mutation, by crossing with Cre transgenic animals. At the same time crossing with FLP expressing mice removed the selection marker and converted the hypomorphic allele to wild type.

A Selection for the marker and

Gene of interest Targeting vector

hypomorhic alleles

Targeted (hypomorphic) allele

H om ologous recom bination

loxP loxP

T ransient expression o f C re recom binase in E S cells

G eneration o f nâce w ith the hypom orphic allele

V

C re excision in situ

Null mutation allele

loxP

H eterologous Knockout ES Knockout mice

B Conditional knockout

Gene of interest Targeting vector

Targeted allele

H om ologous recom bination

T

Conditional null allele

G eneration o f m ice

T

C ross with tissue specific C R E transgenic mice

Tissue specific knockout mice

Mice with reduced levels of the gene in specific tissues only

Restoration of gene expression in specific tissue only

Figure 1.9: A) Strategy for excision o f the selectable m arker an d /o r generation o f hypom orphic alleles. B) Strategy for conditional gene targeting. See text for details.