Figure 7: Proof-of-principle TRiPAdeno vector production using the apoptotic transgene Bax.
(a) Representative phase or fluorescent microscopy images of HEK293T or HEK293T.TRiP cell cultures in the first amplification phase post-inoculation with Adeno-CMV-tbsGFP or Adeno-CMV-tbsBax-iGFP vectors produced in HEK293T.TRiP cells (black scale bar=100 μm). (b) Vector stocks from the final round of amplification/passaging were titrated on HeLa cells and HeLa-TRAP cells. Data are mean average values±s.d. [log10-transformed data] (n=3); *P<5.4 × 10−4, **P<1.5 × 10−4 [Welch’s t-test]. (c) Vector stocks from the final round of amplification/passaging were titrated by qPCR of purified vDNA using a GFP primer/probe set. Data are mean average values±s.d. [log10-transformed data] (n=3); *P<3.1 × 10−2, **P<6.7 × 10−8 [Welch’s t-test]. (d) Vector stocks from the final round of amplification/passaging were used to transduce HeLa and HeLa-TRAP cells at an MOI ∼5, and transduced cell lysates analysed for functional expression of transgene cassettes by immunoblotting to Cleaved-PARP (induced by Bax over-expression), Bax, GFP and GAPDH (molecular weight marker in kilodaltons). Triplicate Adenoviral vector stocks were derived and amplified/passaged from three independent recombination steps; qPCR and immuoblotting were each repeated twice.
