Over the past few years, strategies have been developed for chromosome engineering in ES cells. Such approaches have been used to design novel chromosomal variants, or to mimic altered chromosomes associated with human disease or metastasis (15). Such approaches rely on the sequential targeting of two loxP sites, either in trans (i.e., to different chromosomes) or in cis (some distance apart on the same chromosome), followed by the transient expression of the Cre protein in order to mediate the site-specific recombination event between the loxP sites, leading to the formation of the new chromosomal variant (15,16) (Fig. 3). In this case, usually three ES cell electroporations and resulting screening for the alteration are required: the two end points are targeted separately, and are followed by the introduction of the recombinase, which will mediate the recombination event between them.
This type of strategy can be used to create almost all cataloged forms of chromosomal aberration. Additionally, application of this technology could allow the creation of multiple large-scale chromosomal alterations, for example, a set of nested hemizygous deletions (also known as deficiencies) covering an entire chromosome. These could then be used to reveal novel tumor suppressor genes or functional haploinsufficiencies mapping within the deleted DNA. If a panel of deficiencies is available, screens for interesting phenotypes can be carried out either in culture, or in mice, the latter being particularly amenable to ES cell ^ tetraploid embryo aggregation (17,18) as a means of creating completely ES cell-derived embryos, therefore bypassing the germline for accessing embryonic phenotypes (19).
Fig. 2. Simple gene alterations—introducing a subtle change into a gene of interest. In this example, a point mutation, small deletion, or domain swap (*) is introduced into an exon of a gene using a relacement type vector, containing a floxed neomycin resistance cassette. Transient expression of Cre results in removal of the region between the loxP sites, and as a result the neo selection cassette is removed, thereby rendering the genomic structure identical to wt with the exception of the introducted change and the single loxP site remaining in the first intron.
Since a recombination event between two loxP sites some distance apart or positioned on different chromosomes, is relatively rare, presumably because of physical constraints, a reflection of chromosome architecture and decreased proximity, such strategies are designed incorporating a binary positive selection system that is only activated after a successful recombination recreates the cassette. Thus, the desired recombination event will reconstruct the selectable genetic marker, from two silent portions placed adjacent to each of the loxP sites. The most commonly used selection systems include the reconstitution of a human hypoxanthine phosphoribosyltransferase (HPRT) minigene (16-20), or the juxtaposition of a strong promoter upstream of a selectable marker.
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