Schematic representation of the screening used to identify integration in cytokine-selective inducible loci.

The Rosaβgeo retroviral gene trap contains a splice acceptor (SA) 5′ to a promotorless fusion reporter gene (geo), which when integrated, generates a fusion transcript between the reporter gene and the trapped gene. The Geo protein will be produced from the ATG of the trapped gene or at least from the ATG in the GEO sequence. TF-1 cells were infected with the pRosaβgeo and diluted in 96-well plates under G418 selection for 3 weeks. Surviving clones were expanded, plated in duplicate and submitted to a proliferative assay with different cytokines: GM-CSF, IL-3, or IL-5, in the absence or presence of G418 (1 mg/mL). ○ cells that do not proliferate • growing cells. Clones of type X from GM-CSF–, IL-3–, or IL-5–cultured and infected cells and selected for survival in the presence of G418, and the cytokine present at the time of infection, exhibit a differential cytokine response, ie, if cultured and selected in GM-CSF, it remains sensitive to G418 in the presence of IL-5 and IL-3, whereas it is G418 resistant in the presence of GM-CSF. This indicates that neither IL-3 nor IL-5 induces transcription of a chimeric transcript, whereas GM-CSF does. Clones of type Y do not present a differential cytokine response based on the acquisition of G418 resistance. It acquires G418 resistance in the presence of all the tested cytokines. It corresponds to integration into either housekeeping genes or nonspecifically cytokine-modulated genes in this assay. Clones of type Z correspond to cells that do not proliferate in the presence of the tested cytokine, reflecting a cytokine-response heterogeneity of the cell line. These clones were not studied further.