Figure 2.

Cloning and rescue of recombinant adenoviral vectors. (A) Modular assembly of adenovirus genomes. Modifications need to be introduced into individual modular, before the final assembly. The figure above shows the modification of adenovirus eerily gene region (E1, E2 and E3). (B) Direct cloning of complete adenovirus genomes from purified virions or infected cells and further modification of adenovirus genomes by homologous recombination. For the adenovirus genome engineering, ampicillin-mediated positive selection is used insert the selection marker cassette (SM, ccdB-Amp) in the first step (i). Then, the counter-selection marker containing adenovirus plasmids are treated with restriction enzymes to linearize the plasmid (ii). Finally the linearized adenovirus genome containing plasmid, and the insert flanked by homologous arms to the target region of the adenoviral genome are co-electroporated into the E. coli strain GB05-dir, in which the RecET recombinase is induced with L arabinose to express, enabling linear-linear homologous recombination to replace the ccdB-Amp cassette with aimed insert. Afterwards, the recombinant adenoviral vector can be reconstituted by transfection of the linearized plasmid into the producer cell line (such as HEK 293 cells) for further applications. Created partly with Biorender.com.