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for internal deletions [20], and can comprise ~90% of isolates in end-deletions made with certain BAC clones (PKC unpublished observations).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.