**1.7 Simultaneous insertion of reporter gene & truncation of DNA from BAC ends**

The two separate steps of introducing the EGFP reporter gene and truncating far upstream sequences of the gene of interest in the BAC can be done simultaneously using lox-Tn10 transposons (14). A basal promoter-containing EGFP gene was designed to serve as an enhancer-trap, and placed in front of the loxP arrowhead such that the cassette was retained after the end-deletion of the BAC DNA [see upper panel of Figure 5 for enhancer-trap transposon]. Progressive truncations from one end of the genomic insert with this enhancertrap transposon generated a library of BAC DNA molecules that differ in the position of the enhancer-trap cassette with respect to the start site of transcription of the gene. The collection of well characterized BAC DNA can be introduced individually into zebrafish embryos for expression of the gene in specific tissues (14).

Such enhancer-trap containing BACs can be re-fitted again at the opposite end with DNA cassettes carrying repeat ends of the vertebrate transposon Tol2 as described below.

Fig. 5. Legend: Schematic representation of deletion formation in starting BAC APPb EGFP

enhancer-trap by pTn*lox511*-iTol2kan: Panel A shows generation of starting BAC APPb EGFP enhancer-trap by insertion of the enhancer-trap transposon (inverted triangle), and the location of APPb gene within the BAC clone. Panel B: Shown schematically is the insertion of the Tn*lox511*-iTol2kan transposon into the enhancer-trap APPb BAC DNA. The thick black arrows represent loxP and lox511 sites (discontinuous arrow). DNA cassettes in front of the loxP or lox511 arrows are retained after the loxP-loxP or lox511-lox511 recombinations by Cre. The enhancer-trap DNA cassette is positioned in front of the loxP arrow (panel A), while the iTol2 cassette is located in front of the lox511 arrow. The colored arrowheads pointing outward in the iTol2 cassette correspond to the 200 bp inverted repeat end R and 150 bp inverted repeat end L of the Tol2 transposon [see Suster et al 2009 ref (42)]. The kanamycin resistance gene is between the Tol2 inverted repeat ends.
