Figure 2

Schematic representation of the novel negative selection strategy. a Schematic representation of a conditionally cytotoxic gene cassette composed of an SA site, an IRES sequence, a DT-A gene and a polyA sequence. This cassette, when placed upstream of the 5′ arm of an exon-trapping targeting vector, is expected to function as a negative selection marker. b Schematic representation of the impact of the negative selection cassette on integration events. When an exon-trap vector integrates non-homologously into a gene-coding region, the cells are capable of acquiring drug resistance (i, left panel). In contrast, when an exon-trap vector possesses the negative selection cassette (ExTraPANS vector), the upstream SA site would trap the splicing from the upstream exon (grey box) to allow DT-A gene expression, thereby killing random integrants (i, right panel). On the other hand, the presence of the negative selection cassette is expected not to affect homologous recombination-mediated targeted integration (ii). Abbreviations are as in Figure 1.