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

Abstract

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.

Figure 1

Schematic diagram for replacing loxP with lox66 in BACs. A set of mutant lox sites is shown in the top panel, with the mutant nucleotides shown in green. The inverted repeats are underlined. The bottom panel sketches our strategy to convert a wild type loxP to a lox66 mutant site in BACs while making end-deletions of genomic DNA insert from loxP end.

Figure 2

Schematic representation of the two lox66 transposons constructed. Both use the same framework of markerless transposons with loxP or lox511 [20,7]. The enhancer-trap is adapted from [2].

Figure 3

FIGE analysis of BAC DNA isolated from deletions generated with lox66 transposons: Panel A: The BAC DNA was digested with Not I prior to FIGE. Lane 1 shows DNA from starting BAC-C. Lanes 3-5 and 7-11 display DNA from BAC deletions generated by Cre-recombination of lox66 with loxP using transposons pTnLox66(B)markerless and pTnLox66(B)markerless Enhancer-Trap transposon, respectively. Lane 6 shows DNA from a loxP-Cre independent internal deletion. Lane 2 shows a 5 kb ladder. The vector DNA bands generated with Not I a, b, c, are indicated by the arrows to the left. Size of vector bands b and c are consistent with loxP-Cre dependent recombinations, while vector band a arises from starting BAC-C or from an internal deletion in BAC-C. DNA from BAC deletions made with pTnLox66(B)markerless transposon generated the Not I vector band of the expected size (~6.6 kb) shown in lanes 3-5 of Figure 3 (marked by arrow c). The BAC deletion shown in lane 6 arises from an internal deletion in the genomic insert DNA, and is independent of lox-Cre recombination. The vector DNA band upon Not I digestion of this clone is 10.6 kb in size, and is identical to that of starting BAC clone C (displayed in lane 1, Figure 3A and marked by arrow a). This vector DNA band serves as a characteristic identifying feature for internal deletions [20], and can comprise ~90% of isolates in end-deletions made with certain BAC clones (PKC unpublished observations). These arise due to recombinogenic sites in insert DNA (discussed in [20]). Panel B: A schematic representation of the Not I sites in pTARBAC2.1 vector DNA in BAC clones is shown. Panel C: Location of ends of lox66 substituted deletion clones in lanes7-10 on zebrafish chromosome 9 is indicated. These were obtained by BLAST analyses of the BAC end sequences derived with Seq 1 primer [Additional File 1] with the zebrafish genome sequence.

Figure 4

BAC deletions generated with lox66 transposons pTnLox66(B)markerless and pTnLox66(B)markerless Enhancer-Trap sequenced with Primers Seq 4-compliment and LF8-compliment respectively. Top Panel: Sequence of BAC-C deletions 3, 4, 5, is shown. The four restriction enzyme sites (4 RE sites) indicated in the schematic representation are Not I, Asc I, Pme I and Pac I, and correspond to sites in pTnLox66(B)markerless transposon shown in Figure 2 top panel. These sites are highlighted by different colors in the sequence presented. Bottom Panel: Sequence of BAC-D deletions 7-11 obtained with pTnLox66(B)markerless Enhancer-Trap transposon is shown. The Pac I site is highlighted in color. Sequence complimentary to the lox66 site is colored and underlined in both panels. Sequencing in the opposite direction with transposon end-based primer Seq 1 in deletion clones 3-5 and 7-11 indicates zebrafish DNA which BLASTs to chromosome 9 (not shown). The locations of these sequences on Chr 9 are consistent with end-points of the BAC deletions expected from their sizes on the FIGE gel shown in Figure 3.

Figure 5

A schematic representation of the three Cre-recombination steps in the end-deletion/lox66 substitution process. Step 1 shows the end-deletion of genomic insert DNA by the transposed lox66 site recombining with the loxP endogenous to the BAC clone. Step 2 illustrates co-integrate formation between lox66 APPb BAC and P1 phage DNA carrying loxP. Step 3 shows regeneration of circular lox66 BAC from the linear DNA in the phage head after infection of fresh bacteria. The lox511 site at the other end of genomic insert DNA is omitted for clarity purposes, as it does not play any role in Cre recombinations with either lox66 or loxP. Note that the relative position of the two lox sites in the linear DNA inside the phage head determines whether lox66 or loxP is retained in the BAC. This in turn is determined by the location of the "pac site" in the co-integrate shown in Step 2 (see [16,20], for detailed discussion).