Identification of transiently expressed genes by gene trap mutagenesis and site-specific recombination. (a) U3Cre gene trap activation from integrations in genes induced by IL-3 withdrawal excises the Tkneo fusion gene, which is flanked by loxP sites from the reporter plasmid pgklxTkneoIL3. This places the IL-3 cDNA immediately downstream of the pgk promoter and enables its expression. This converts FLOXIL3 cells to factor independence. Cre, cre recombinase; Pol II, RNA polymerase II; pgk, phosphoglycerate kinase promoter; lx, loxP target sequence for Cre recombinase; Tkneo, HSV-thymidine-kinase/neomycin-phosphotransferase fusion gene; pA, bovine growth hormone polyadenylation sequence; IL-3, murine interleukin-3 cDNA. (b) Selection strategy for genes induced by IL-3 withdrawal. U3Cre-infected FLOXIL3 cells were first selected in G418 to eliminate integrations into constitutively expressed genes. The G418-resistant cells were then plated into semisolid (agar) cultures in the absence of IL-3. Autonomous colonies were picked and expanded for further analysis.