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Background  Chromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps, unreliable. Gene targeting to a pre-determined site on the chromosome is likely to alleviate the problem.    Findings  A general procedure to replace the  loxP  site located at one end of genomic DNA inserts in BACs with  lox66  is described. Truncating insert DNA from the  loxP  end with a Tn10 transposon carrying a  lox66  site simultaneously substitutes the  loxP  with a  lox66  sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of  loxP  with  lox66  or  lox71  was found to be as efficient as another  loxP  site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a  lox66  transposon occurred at no more than 20% the efficiency observed with a  loxP  transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical  lox -sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed.    Conclusions  The  loxP/lox66  replacement procedure should allow targeting BACs to a pre-positioned  lox71  site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.

Replacing the wild type loxP site in BACs from the public domain with lox66 using a lox66 transposon