Figure 3: Repression of multicistronic transgene cassettes using the TRAP-tbs configuration.
(a) Bicistronic reporter plasmids encoding Firefly luciferase (Luc; ORF1 position) and green fluorescent protein (GFP; ORF2 position); ORF2 being dependent on cap-independent translation. The constructs vary in position and number of tbs sequences controlling the ORFs. (b) HEK293T cells were transfected with the bicistronic plasmids±TRAP, and a Renilla Luciferase plasmid was co-transfected in all conditions to allow normalization of FireFly luciferase activity. Duplicate sets of cells were either analysed for normalized luciferase activity in cell lysates (left y-axis; yellow bars) or live cells analysed for GFP expression by flow cytometry (right axis; green bars). All data are mean average values±s.d. [log10-transformed data] (n=3); *P<1.5 × 10−7, **P<1.4 × 10−5 [Welch’s t-test]. (CMVp, Cytomegalovirus promoter; tbs, TRAP-binding sequence; IRES, internal ribosomal entry site (encephalomyocarditis virus); polyA, polyadenylation signal). Results are representative of two independent experiments.
