Figure 3

Panel A: FIGE analysis of enhancer-trap BACs with mutated intron 1 enhancer from different libraries. Panel B: FIGE analysis of clone DNA from the library generated by inserting Tnlox511-iTol2kan at the lox511 end of the enhancer-trap BAC in lane 12, Panel A (marked by yellow arrowhead). Panel C: EGFP fluorescence from transient expression in neurons (marked by the pink arrowheads) of zebrafish injected with mutated but functional intron enhancer-trap BAC with intact upstream DNA, Panel D: EGFP expression in notochord (indicated with pink arrowheads) from injecting enhancer trap BAC with mutated but functional intron enhancer and with 31 kb upstream DNA deleted (such as clone in lane 21, panel B, red arrowhead) taken from the same library as the BAC used for Panel C. Panel E: EGFP fluorescence in neurons (marked by the pink arrowhead) from a F2 transgenic zebrafish line obtained from the enhancer-trap BAC shown in lane 11 of Panel B (marked by blue arrowhead). The mutated but functional intron enhancer used was deleted for GATA3, OCT1 and the CT-repeat element (clone shown schematically in row 8, Panel B of Figure 2). Additional examples of germline transgenic fish with slightly smaller enhancer-trap BAC transgenes, but containing the upstream ~31 kb sequence, are shown in Figure 6 panels A and B of reference [23]. The BAC vector DNA band from Not I digestion is shown by the black arrowhead to the left of panels A and B. Lane 2, Panel B, contains the same DNA as lane 12, Panel A. Lanes 3–21 in panel B do not have this band because insertion of Tnlox511-iTol2kan at the lox511 end of BAC DNA and subsequent lox511-lox511 deletion eliminates the Not I site at that end [23].