Fig 1.
Overview of the Adenobuilder system.
Segments of the Ad5 genome were individually amplified and cloned into high-copy plasmids. Each of the five central “building blocks” contains unique sequences at either end (represented by distinct colors) that create a 15–20 bp overlap with the respective neighboring block. Each block is flanked by recognition sites for the restriction enzyme BstBI. Following digestion, the unpurified plasmid fragments are directly added to an isothermal assembly reaction and then delivered to packaging cells via electroporation. This system thus facilitates the generation of infectious adenovirus particles from plasmids in three steps that can be carried out in under two hours.
Fig 2.
Partitioning of the Ad5 genome.
The cloned blocks 1–7 are aligned to a map of the Ad5 genome. Protein-coding genes are indicated in red, with arrows indicating the strand that is transcribed. The inverted terminal repeats (ITRs), which define the left and right ends of the adenovirus episome, and virus-associated RNAs (VA RNA) are shown. The early and late transcriptional units are marked by green and black arrows, respectively.
Fig 3.
Seamless ligation of adjacent genomic blocks.
The free ends created by BstBI are initially processed by the heat-labile T5 exonuclease, creating an extended 3’ overhang at each end. When complementary strands anneal, the ends are trimmed, filled in and ligated. The thermostable BstBI enzyme remains active during the assembly process and thus prevents re-ligation of the cut ends. The compatible ends of Blocks 1 and 2 are shown; the other junctions are simultaneously processed in the same manner.
Fig 4.
Reassembly and packaging of infectious Ad5.
A. Equimolar amounts of each of the seven Adenobuilder plasmids were digested with BstBI and then incubated for an additional 60 min with (+) and without (-) the assembly enzyme mix. Aliquots containing 660 ng total DNA were assessed on a 0.8% agarose gel. Size markers (in kb) are shown on the left; the bands corresponding to the vector backbone and each of the excised blocks is indicated at right. B. Unassembled (upper panel) or assembled (lower panel) blocks were delivered to HEK293 cells by electroporation. Images were captured at 36 h, at 20X magnification. Arrows indicate representative rounded cells. Scale bar = 200 μm. C. HEK293 cells were infected with 10 μl of the primary lysate, derived directly from cells that had been electroporated (upper panel), or with 1 μl of lysate obtained after a single round of amplification (lower panel). Viral titers were determined by detection of the Ad5 hexon protein (green), which is expressed late in the viral life cycle. Magnification, 10X. Scale bar = 400 μm. D. Genomic DNAs from the reference Ad5 stock (Ref) and assembled Ad5 (AB) were compared by restriction digest. One μg of DNA from each sample was digested with EcoRV or HindIII. Fragments of the indicated sizes were resolved on 0.8% agarose. E. A viral particle in a primary, unpurified lysate was visualized by electron microscopy after negative staining, Magnification, 135000X. Scale bar = 100 nm.
Fig 5.
Two-step derivation of mutations in E1B and E4.
A. The mutant constructs were first amplified with two partially overlapping primers designed to incorporate the desired alteration (shown in green), using the corresponding wild type block as a template. The input template DNA was then eliminated by digestion with the restriction enzyme DpnI. In the second step, the PCR-derived plasmid was circularized in an assembly reaction, which merged and sealed the overlapping ends. B. Oligonucleotide pairs that were used to generate the mutants are illustrated. The mutant block 1-derived constructs were E1B19K-null, E1B55K-null or modified to express an endogenous E1B55K protein with a c-terminal FLAG epitope. Similarly, the E4ORF3 open reading frame was disrupted by a mutation at the initiation codon or the introduction of a c-terminal FLAG tag. The targeted codons are boxed, single base mutations are shown in red. At positions where the initiation codon of the target protein overlapped with a codon for a different protein, base substitutions were selected so that the mutation would be silent with respect to the second open reading frame. C. hTERT-RPE1 cells were infected with the synthetic Ad5 virus and the E1B mutant viruses generated in this study. Cells were lysed 16 h post-infection, and assessed by immunoblot with antibodies against p53 and the FLAG epitope, as indicated. D. hTERT-RPE1 cells were uninfected (no virus) or infected with wild type Ad5 and the E4 mutants. For all viruses, the MOI was 100. GAPDH was probed as a loading control.
Fig 6.
Modifications to blocks 1, 6 and 7 facilitate the incorporation of large transgenic elements into recombinant Ad5 vectors.
A, DNA cassettes up to 3 kb in size can be directly cloned into a polylinker in the modified block pAd5-B1ΔE1-MCS. Still larger transgenic DNAs can be incorporated into assembled viruses with the use of plasmids pAd5-B6ΔE3 and/or pAd5-B7ΔE4. B, successively larger transgenes can be introduced with the deletion of E1, E3 and E4. Used together, these three modified blocks thus allow the customized assembly of recombinant viruses capable of delivering transgenic elements up to 8.5 kb in size.