Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Abstract

Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene-harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, single plasmid-based adenoviral vector systems were constructed where a unique PmeI site was located at the position for insertion of the exogenous gene. The polymerase chain reaction (PCR) amplified transgene could be cloned into PmeI-linearized starting plasmids using one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was termed restriction assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded starting plasmid pKAd5f11pABR-EPG. The modified fiber gene, amplified by overlap extension PCR, could be used to substitute the original fiber in pKAd5f11pABR-EPG to generate an adenoviral plasmid with a new fiber by restriction assembly. On the other hand, pKAd5f11pABR-EPG was also a starting adenoviral plasmid for expressing other transgenes.In conclusion, easy-to-use and upgradable adenoviral vector systems are introduced here, which offer extensive versatility and can serve as a basic platform and functional component library for the synthetic biology of adenoviral vectors.

No Genus-Specific Gene Is Essential for the Replication of Fowl Adenovirus 4 in Chicken LMH Cells

“…In some cases, point mutations, which led to early termination of translation or frameshift mutations, were used to block the generation of functional gene products. The following procedure was utilized to generate FAdV-4 mutants: a DNA fragment was excised from the adenoviral plasmid by restriction digestion and used to construct an intermediate plasmid, in which more unique restriction sites could be used for site-directed modification; mutations were introduced into the intermediate plasmid; and finally, the modified intermediate plasmid was restored to the original adenoviral plasmid to generate a new one ( 26 , 27 ). The start adenoviral plasmid pKFAV4-CX19A had the deletions of ORF1, ORF1B, and ORF2 at the left end and ORF19A at the right end of the FAdV-4 genome, and it also carried the insertion of CMV promoter (CMVp)-controlled mCherry expression cassette ( 24 ).…”

Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector

“…Conclusively, based on others’ work and our experience [ 11 , 21 , 27 , 44 , 47 ], we present restriction-assembly as an all-purpose method for the construction of novel adenovirus vectors or modification of existing adenovector systems. This strategy possesses many advantages and will benefit the synthetic biology of adenoviruses.…”

“…Finally, the restriction-assembly can be used to load a transgene to an adenovirus vector [ 47 ]. The adenovirus plasmids constructed based on the restriction-assembly principle will contain unique or dual cutter restriction sites in the transgene cloning region.…”

Fiber modifications enable fowl adenovirus 4 vectors to transduce human cells

“…pMD‐FAV4F1CDR was digested with Kpn I/ Eco RV to recover fragment containing fibers (2997 bp), pKFAV4GFP was digested with Mau BI/ Sbf I to recover the large fragment (43,078 bp) and these two fragments were mixed for DNA assembly to generate adenoviral plasmid pKFAV4F1CDR‐GFP (restriction‐assembly; see Supporting information, Figure S2 ). 34 , 35 To insert RGD4C into the fiber2 CD loop, overlap extension PCR was performed to amplify F1‐F2CDR fragment (3007 bp), which was inserted at the Mau BI/ Sbf I sites in pKFAV4GFP to generate pKFAV4F2CDR‐GFP through DNA assembly (see Supporting information, Figure S3 ). The intermediate plasmid‐based system was employed for replacing the CMV promoter with human EF1a promoter to generate adenoviral plasmid pKFAV4‐EG.…”