Journal: Virology Journal

Article Title: Characterization of human adenovirus 35 and derivation of complex vectors

doi: 10.1186/1743-422X-7-276

Figure Lengend Snippet: Effect of E3 deletions on fiber transcription and virus fitness . (A) Northern blot of 24 hpi total RNA from 293-ORF6 cells infected with wild type Ad35 (wt), E1-deleted rAd35 (d8), or rAd35 d8 constructs with the E3 deletions noted in panel B. The blot was hybridized with a fiber (top) or pIX probe (bottom); the fiber and pIX transcripts are labeled. Numbers on the right denote migration of RNA size standards (kilobases). (B) Schematic of the Ad35 E3 region and E3 deletions (not to scale). The coordinates of the nucleotides that form the deletion junctions are shown above the lines. L4 poly(A), E3 poly(A) = L4 and E3 polyadenylation hexanucleotide signals, respectively. (C) and (D) Viral vector growth competitions. rAd35 d8 E1-deleted vector was mixed with (C) rAd35 d8, E3(X)-deleted vector or with (D) rAd35 d8, E3(HE)-deleted vector. Relative change in genome amounts was determined by DNA restriction fragment analysis of the input mixture of viruses and each serial passage. DNA restriction fragment analysis uniquely identified each virus genome, which is indicated to the left of the gel and their fragment sizes in bp on the right. I = input mixed viruses used for initial infection; M = mock infected cells; P1 = initial infection; P4 = fourth passage; a, b, c = replicates. Restriction enzymes EcoRV and BlpI were used in panels C and D respectively.

Article Snippet: To distinguish the E3 wild type and E3 deletion X and wild type from deletion HE, the viral genomes were restricted with EcoRV and BlpI endonucleases (New England BioLabs), respectively, before being resolved on a 0.8% agarose gel using 0.5% TBE buffer.

Techniques: Northern Blot, Infection, Construct, Labeling, Migration, Plasmid Preparation