FIG. 1.

Generation of recombinant cancer-targetable Ad5 vectors. (A) Overview of viral modification and production using recombineering. (1) Design of the selection cassette, (2) temperature-induced (42°C) recombineering of the selection cassette into Escherichia coli strain SW102 by electroporation, (3) recombineering of the target sequence by electroporation, (4) verification of the correct clone by sequencing, purification of DNA by maxiprep, and generation of P1 virus stocks in permissive T-REx-293 cells, (5) propagation of high titer P2 stocks, (6) purification of viral particles by CsCl gradient ultracentrifugation and dialysis, and (7) titration by microBCA assay. (B) Amino acid sequence alterations within the Ad5 fiber knob domain. Peptide sequences GE11, YHWYGYTPQNVI; M*, MQLPLAT; LS, LSPPRYP; and scramble, LMTLAQP, were genetically inserted into fiber knob HI loop after Thr541. For the purpose of native hCAR-binding ablation, KO1 mutation (S408E, P409A) was introduced into the AB loop. (C) The inserted peptides (shown in green) GE11, M*, LS, scramble, and the HI loop (magenta) are highlighted. Inset: side view of the fiber knob, showing native (gray) or mutated hCAR-binding site (yellow). EGFR, epidermal growth factor receptor; FGFR1, fibroblast growth factor receptor 1; hCAR, coxsackie and adenovirus receptor; KO1, hCAR-binding mutation. (D) Verification of fiber integrity by Western blotting. Color images available online at www.liebertpub.com/hum