Targeted integrations

Abstract

Abstract Apart from generating deletions, are recombinase also mediates the reverse, integrative reaction by recombining loxP-containing, incoming vectors with a transgene or endogenous sequences harboring loxP sites in the genome. Thus, the capacity of the Cre/loxP system for site specific integration could be used as an alternative for homologous recombination to generate secondary modifications in loxP containing loci in cultured cells. Possible applications of loxP-mediated integration could include, for example, the analysis of different promoter regions into the same genomic site for gene expression studies or the replacement of different mutated exons in structure/function studies. For targeted integration, the loxP containing integration vector must be introduced together with a vector for transient Cre expression, ensuring only limited recombinase activity, to avoid the subsequent excision of the integrated, loxP-flanked DNA segment. In plant cells it has been shown that the equilibrium of Cre¬ mediated inversion can be shifted to one side by using a pair of two different mutant lox sites (42). Certainly this technique will be also used in future to stabilize the products of Cre-mediated integration in ES cells by preventing reexcision. A circular vector containing a single loxP site will be integrated completely (Figure 8), whereas from a linear vector with two loxP sites in the same orientation only the loxP-flanked region will be inserted, probably after extrachromosomal resolution of the floxed segment into a circle with a single loxP site (Figure 8).