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

Abstract

BackgroundChromatin 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.FindingsA 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.ConclusionsThe 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.

Functionalizing Bacterial Artificial Chromosomes with Transposons to Explore Gene Regulation

“…For example, cross recombination between loxP and lox511 has been reported to occur at efficiencies ranging from 5 to 100 % under those experimental conditions that express Cre constitutively (33,(35)(36)(37)(38). We have not observed such cross-recombination, and high levels of stringency in vivo in recombining identical lox sites (21,24), or lox sites with at least identical spacers (39), have been achieved with Cre protein expressed from its native source namely, a phage P1 infection (21,24,39). Depending on whether a loxP or a lox511 transposon is used, truncations from the corresponding lox-end can be made readily and exclusively with high stringency [shown schematically in Figure 4].…”

Long range gene-regulatory sequences identified by transgenic expression of bacterially-engineered enhancer-trap BACs in zebrafish

“…Previous studies had reported recombination between loxP and lox511 to occur at efficiencies ranging from 5 to 100% under experimental conditions that express Cre constitutively [54][55][56][57][58][59]. Such cross-recombination was not observed when Cre protein was expressed in a phage P1 infection in vivo [49,60,61], and high levels of stringency in recombining identical lox sites [49,60], or lox sites with at least identical spacers was achieved [61]. Truncations of genomic DNA in a BAC from either end are not only efficient, but are specific for that end.…”

Trapping Enhancers by Transgenic Expression of BACs Engineered in Bacteria with loxP Transposons

“…Previous studies had reported recombination between loxP and lox511 to occur at efficiencies ranging from 5 to 100% under experimental conditions that express Cre constitutively [70][71][72][73][74][75]. Such cross-recombination was not observed when Cre protein was expressed in a phage P1 infection in vivo [65,76,77], and high levels of stringency in recombining identical lox sites [65,76], or lox sites with at least identical spacers was achieved [77]. Truncations of genomic DNA in a BAC from either end are not only efficient, but are highly specific for that end.…”