FIG. 5.

Construction of recombinant adenoviruses with hTfR-targeting motifs in the HI loop. A series of HI loop-modified shuttle plasmids were generated (pBS/BxHI; see Materials and Methods). These shuttles were cut with BamHI and NotI, and the 4.6-kb fragment was cotransformed with SwaI-cut pTG3602RSVGFP/SwaI into E. coli BJ5183. Appropriate recombination resulted in full-length Ad5 genomes with a GFP expression cassette in the E1 region and B1, B2, etc., motifs in the HI loop. The recombinant plasmids were restricted with PacI and transfected into HEK 293 cells, and virus was harvested and propagated upon finding evidence of cytopathic effect.