Panel A: Schematic representation of bio-informatically predicted transcription factor binding sites in the intron 1 enhancer (IE) within the enhancer-trap (taken from Additional file 2: Figure S2 of reference ), highlighted by the colored letters. Panel B: Mutated enhancer-trap BACs used in this study: Mutations and deletions, represented schematically with dotted lines of same color in place of the letters, were engineered into the intron enhancer (IE) in the small enhancer-trap transposon plasmid at the sites marked by CT-repeat, SOX5, E4BP4, XFD1, OCT1, and GATA3. Each mutated enhancer-trap transposon was inserted into appb BACs C and D to generate the enhancer-trap BACs indicated on the side of the corresponding mutation. For example the blue dashed line in the first row indicates a deletion of the CT-repeat sequence in enhancer-trap (shown as dotted blue line), and the BACs containing this mutation are Δ2C, Δ76C and Δ51D. Because SOX5 and E4BP4 sites overlapped, only point mutations were introduced into the SOX5 site to obtain the plasmid with wild type E4BP4 and point mutation in SOX5 and deletion of CT-repeat, shown in second row Panel B. Enhancer-trap BACs with these mutations are Δ5C Δ14C, Δ17D Δ21D. Row 7 shows the wild type enhancer-trap with Δ94C, and Δ74D as representatives. The results of expressions of these BACs are summarized in Table 1. The last enhancer-trap BAC in Panel B (row 8) has enhancer-trap deleted for CT-repeat, OCT1 and GATA3. This BAC is also deleted from the lox511 end of BAC with the Tnlox511-iTol2kan to make the germline transgenic zebrafish shown in Figure 3, Panel E. The bent lines on both ends of the BAC represent end-truncations by the transposons, and illustrate the location of the particular transposon end preserved after either the loxP-loxP or lox511-lox511 deletions mediated by Cre protein, respectively.
Shakes et al. BMC Genomics 2012 13:451 doi:10.1186/1471-2164-13-451