**1.8 BAC transgenesis in zebrafish and mice using Tol2**

BACs have been integrated into the germlines of zebrafish and mice using the vertebrate transposon Tol2 system (42). Tol2 ends, in the inverted orientation and flanking a 1 kb spacer DNA (iTol2), were introduced into the BAC DNA within the bacterial host using recombination of homologous sequences between the targeting vector and the genomic insert. This approach used to introduce the iTol2 cassette can have unintended consequences: other sequence repeats existing in BACs from vertebrate DNA libraries were likely to rearrange as well. This would complicate the introduction of iTol2 cassettes at best, and were likely to present major challenges in BACs spanning chromosomal loci that contained highly repetitive DNA, such as the Npr3 gene locus analyzed earlier (16). Therefore a simpler and more flexible system was developed using our Tn10 transposon approach to deliver iTol2 ends into BACs. These iTol2-end containing BACs are suitable for transgenesis into zebrafish or mouse embryos, and have recently been reported (15).

The iTol2 DNA cassette was placed in front of a loxP or lox511-Tn10 transposon (15). Progressive truncations from an end of the genomic insert with this iTol2-Tn10 transposon generated a library of BAC DNA molecules that differed in the position of the iTol2 cassette with respect to upstream regulatory elements of the Amyloid Precursor Protein (APPb) gene in the BAC. The collection of well characterized BAC DNA was introduced into zebrafish embryos individually for expression of EGFP in neurons (Figure 6).

Fig. 6. Legend: Panel A: FIGE analysis of an array of APPb BACs with EGFP containing Enhancer-trap at loxP end and iTol2kan cassette at lox511 end of DNA insert. The BAC DNA was digested with Not I before FIGE. Note that there is no vector DNA band because the lox511-lox511 deletion with Cre removes both the Not I site in front of the BAC lox511 and the Not I site behind the lox511 in the transposon shown in panel B of Figure 5. Panel B: EGFP fluorescence in Germline transgenic F1 zebrafish obtained from injecting eggs (F0 zebrafish) with the BAC DNA shown by the blue arrowhead in lane 15.

BACs have been integrated into the germlines of zebrafish and mice using the vertebrate transposon Tol2 system (42). Tol2 ends, in the inverted orientation and flanking a 1 kb spacer DNA (iTol2), were introduced into the BAC DNA within the bacterial host using recombination of homologous sequences between the targeting vector and the genomic insert. This approach used to introduce the iTol2 cassette can have unintended consequences: other sequence repeats existing in BACs from vertebrate DNA libraries were likely to rearrange as well. This would complicate the introduction of iTol2 cassettes at best, and were likely to present major challenges in BACs spanning chromosomal loci that contained highly repetitive DNA, such as the Npr3 gene locus analyzed earlier (16). Therefore a simpler and more flexible system was developed using our Tn10 transposon approach to deliver iTol2 ends into BACs. These iTol2-end containing BACs are suitable for

transgenesis into zebrafish or mouse embryos, and have recently been reported (15).

embryos individually for expression of EGFP in neurons (Figure 6).

The iTol2 DNA cassette was placed in front of a loxP or lox511-Tn10 transposon (15). Progressive truncations from an end of the genomic insert with this iTol2-Tn10 transposon generated a library of BAC DNA molecules that differed in the position of the iTol2 cassette with respect to upstream regulatory elements of the Amyloid Precursor Protein (APPb) gene in the BAC. The collection of well characterized BAC DNA was introduced into zebrafish

Fig. 6. Legend: Panel A: FIGE analysis of an array of APPb BACs with EGFP containing Enhancer-trap at loxP end and iTol2kan cassette at lox511 end of DNA insert. The BAC DNA was digested with Not I before FIGE. Note that there is no vector DNA band because the lox511-lox511 deletion with Cre removes both the Not I site in front of the BAC lox511 and the Not I site behind the lox511 in the transposon shown in panel B of Figure 5. Panel B: EGFP fluorescence in Germline transgenic F1 zebrafish obtained from injecting eggs (F0 zebrafish) with the BAC DNA shown by the blue arrowhead in lane 15.

**1.8 BAC transgenesis in zebrafish and mice using Tol2** 
