**2.3 Substitution of Lox sites flanking BAC inserts with Lox66**

In a different application Tn10 transposons carrying 'arm' mutants of loxP, such as lox66, have been used to successfully substitute the loxP site at one end of the genomic insert DNA in a BAC (39). It should be possible also to substitute the lox511 site at the other end of insert DNA in a similar manner using a lox site with an identical 'spacer' as lox511 but carrying a different 'arm' sequence. The usefulness of such substitutions of loxP or lox511 sites in BACs with lox66 is realized from the fact that the integration of loxP plasmids to a loxP site on the chromosome is reversible, with the equilibrium favored for excision. Targeting lox66 substituted BACs specifically to pre-positioned lox71 sites in chromosomes via lox-Cre recombination should be irreversible. Such strategies are likely to be particularly useful in systems such as the zebrafish, where classical "knock-in" technology cannot be used because of genome duplication in an ancestral teleost [see reference (39) for discussion].
