Figure 1.

Engineering and screening of multiple S-TRAIL and luciferase fusions in vitro. (A): Schematic representations of lentiviral transfer vectors bearing IRES-GFP cassettes and encoding various fusions between secreted variant of the pro-apoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (S-TRAIL) and different luciferase proteins. Direct fusion variants: (1) TRAIL-Rluc, (2) TRAIL-Fluc, (3) TRAIL-GpLuc, (4) GpLuc-TRAIL. Variants to test intramolecular spacing: (5) GpLuc-linker 1-TRAIL, (6) GpLuc-linker 2-TRAIL. Variants to test modification of secretion sequence: (7) SGpLuc-Linker 2-TRAIL, (8) SRluc_O-linker 2-TRAIL. To screen the various fusion molecules, 293T cells were transduced with lentiviral vectors encoding the designated fusion variant. Bioluminescence imaging and enzyme-linked immunosorbent assay were performed on conditioned medium from the transduced cells to determine diagnostic luciferase activity or concentration of S-TRAIL, respectively. Therapeutic activity of each variant was determined by luciferase-based assay on human Gli36-EGFRvIII cells 24 hours after incubation with equal volumes of conditioned media from lentiviral transduced 293T cells. Abbreviations: GpL₁TR, GpLuc-linker 1-TRAIL; GpL₂TR, GpLuc-linker 2-TRAIL; GpTR, GpLuc-TRAIL; SGpL₂TR, SGpLuc-Linker 2-TRAIL; SRL_OL₂TR, SRluc_O-linker 2-TRAIL; TRFL, TRAIL-Fluc; TRGp, TRAIL-GpLuc; TRRL, TRAIL-Rluc.