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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Findings</p> <p>A general procedure to replace the <it>loxP </it>site located at one end of genomic DNA inserts in BACs with <it>lox66 </it>is described. Truncating insert DNA from the <it>loxP </it>end with a Tn10 transposon carrying a <it>lox66 </it>site simultaneously substitutes the <it>loxP </it>with a <it>lox66 </it>sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of <it>loxP </it>with <it>lox66 </it>or <it>lox71 </it>was found to be as efficient as another <it>loxP </it>site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a <it>lox66 </it>transposon occurred at no more than 20% the efficiency observed with a <it>loxP </it>transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical <it>lox</it>-sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed.</p> <p>Conclusions</p> <p>The <it>loxP/lox66 </it>replacement procedure should allow targeting BACs to a pre-positioned <it>lox71 </it>site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.</p