Figure 6: Repression of GFP during production of TRiPAdeno-GFP vectors.

(a) The TRiPAdeno system, which requires: vector genome (with tbs-modified transgene(s)), helper functions and a TRAP expression cassette. TRiPAdeno is suited to the production of first/second generation Ad vectors, as well as Gutted (Helper-dependent) and Helper-independent Oncolytic Ad vectors. (b) The tbs-modified adenoviral type 5 shuttle plasmids used in the study, containing the ‘left’ ITR, transgene cassette and adenovirus homology block of 9.2–16.1 map units. Bax was used to exemplify production of a vector expressing high-levels of a toxic gene (see text and Fig. 7). (c) Repression of GFP expression during vector recombination together with the pRAPAd back-bone plasmid in HEK293T cells. Data are mean average values±s.d. [log₁₀-transformed data] (n=3); *P<6.7 × 10⁻⁵ [Welch’s t-test]. (d) Vector stocks generated from (c) were used to transduce either HEK293T or HEK293T.TRiP cells at a multiplicity of infection (MOI) of 0.01, and replicate cultures analysed by flow cytometry at different points post-inoculation during one round of vector amplifcation phase to generate GFP Expression Scores. (Pro, promoter; ITR, inverted terminal repeat; Ψ, packaging signal; tbs, TRAP-binding sequence; ORF, open-reading frame; `i', internal ribosomal entry site (encephalomyocarditis virus); polyA, polyadenylation signal; An, poly-adenides).