Figure 2

Summary of the basic gene trapping strategies. Genomic integration of the gene trap markers is facilitated by transposition. (a) Structure of a putative endogenous target gene. (b) The enhancer traps incorporate a reporter expression cassette driven by a minimal promoter (mP) that only results in reporter gene expression when it is affected by a genomic enhancer element, for example by transposition into a gene. (c) The conventional gene trapping cassettes contain a splice acceptor (SA) followed by a reporter gene and a polyadenylation signal (pA). The reporter is only expressed when transcription starts from the promoter of an endogenous transcription unit. Thus, the expression of the reporter follows the expression pattern of the trapped gene. The GAL4 system is a particularly interesting version of gene or enhancer trapping in Drosophila. Here, GAL4 expression is driven by the trapped regulatory regions of endogenous genes in GAL4 driver lines. Using these driver lines, any protein of interest can be over-expressed or mis-expressed by crossing these lines with others carrying the protein of interest expressed from GAL4 controlled promoter (upstream activator sequence [UAS]). (d) Polyadenylation (poly(A)) traps contain a promoter followed by a reporter gene and a splice donor (SD) site, but they lack a poly(A) signal. Therefore, reporter gene expression depends on splicing to downstream exon(s) of a Pol II transcription unit containing a poly(A) signal. (e) The 'dual tagging' vectors are based on both gene and poly(A) trapping of a targeted transcription unit. (f) The protein trap strategy inserts an artificial exon encoding a reporter into a gene, where the reporter is designed to be incorporated at the protein level into the endogenous gene product. The P element based protein trap (PTT) vector set has been created to tag proteins in all three reading frames with green fluorescent protein (GFP) in Drosophila. (g) Targeted over-expression/mis-expression is a version of the poly(A) trap strategy. Here, a strong promoter (sP) oriented toward the outside of the element is directly followed by a splice donor site. This strategy allows over-expression/mis-expression of truncated or full length endogenous proteins, depending on the site of vector integration. An improved version of this approach is the so-called modular mis-expression system in Drosophila. Here, a GAL4 controlled promoter (UAS) is inserted by the P element into an endogenous transcription unit. This arrangement allows expression of the trapped gene in any arbitrary manner of interest by crossing the carrier line with a GAL4 driver line. E1 to E4, exons 1 to 4; GAGA, GAGA transcription factor (GAF) binding site; ITR, inverted terminal repeat; P, promoter; pA, poly(A).