Gene Trap
It is feasible that gene insertion strategies utilizing loxP sites may gain popularity in certain gene-trap experiments, the goal of such experiments being to identify novel ubiquitous and/or lineage-restricted promoter/enhancer elements or genes.
Using a vector carrying a splice acceptor sequence placed upstream of a reporter gene (such as lacZ or GFP [green fluorescent protein]), different types
Fig. 4. Trapping an endogenous locus with the option of introducing a new gene. Here two electroporation steps are required. The first is used to identify a locus of interest, and the second is used to introduce the new gene into that locus. Two alternative alleles are created on the second electroporation, one doubly drug-resistant, the other singly. Thus, the drug selection will determine which clones can grow. P, promoter driving gene expression in ES cells; SA, splice acceptor; Pgeo, Pgal-neo gene fusion; puro, puromycin resistance cassettes; IRES, internal ribosome entry site.
Fig. 4. Trapping an endogenous locus with the option of introducing a new gene. Here two electroporation steps are required. The first is used to identify a locus of interest, and the second is used to introduce the new gene into that locus. Two alternative alleles are created on the second electroporation, one doubly drug-resistant, the other singly. Thus, the drug selection will determine which clones can grow. P, promoter driving gene expression in ES cells; SA, splice acceptor; Pgeo, Pgal-neo gene fusion; puro, puromycin resistance cassettes; IRES, internal ribosome entry site.
of regulatory or gene sequences can be trapped (31-33). ES cell-chimeric embryos are stained for the histochemical marker to reveal expression domains of the trapped elements (31-33). On the basis of the expression pattern information gained on the trap cell lines, a subset is chosen for further study. If a specially designed trapping vector is used, such as that illustrated in Fig. 4, the trapped locus can be retargeted via loxP sites, and different transgenes can be knocked-in leading to their spatiotemporal expression being governed by the trapped element. Later, the expression can also be abolished by introducing the Cre recombinase into the system.
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